A.P.R.I.L. Effects of Nox and NH3 on Lichen Communities and Urban

Effects of NO and NH on lichen x 3 communities and urban ecosystems

A Pilot Study

A report produced by Imperial College & The Natural History Museum, as partners in the

A.P.R.I.L.

Network for the Department for Environment, Food and Rural Affairs

March 2002

AA..PP..RR..II..LL.. AIR POLLUTION RESEARCH IN LONDON A research network supported by EPSRC, DEFRA & EA

APRIL is a multidisciplinary research network that aims to deliver research projects identified by academics, local and national government and other stakeholders working in urban environments. Through regular seminars, workshops and meetings a large research programme has arisen covering the following major topics: Modelling, Measurements, Meteorology, Natural Environment, Health, Planning, Economic & Social Issues, Transport and Indoor Air Quality. A key element of the research programme is collaboration between many different disciplines and organisations in London and other parts of the UK. By combining elements of each specialist group’s research programme two large consortium proposals have arisen and several more are in preparation. The first, Meteorology and Air Pollution in London’s Environment (MAPLE, measures meteorological and pollution parameters of the air mass coming into London, in and above the city itself and the air mass leaving the city. It involved climatic and pollutant measurements at three sites, Cliffe on the southeastern perimeter of the city, Regents Park in the centre of London and Silwood Park to the West. Campaigns to assess pollution at ground level and in the vertical profile are proposed to provide a greater understanding of the sources of particulates and oxides and nitrogen and their chemistry and dispersion over a large city to specifically inform decisions on the most appropriate abatement strategies to meet London’s air quality objectives and generally to improve scientific understanding of the urban environment. Funding is required.

The second consortium proposal, Dispersion of Air Pollution & Penetration into the Local Environment (DAPPLE), funded by EPSRC, investigates the finer scale pollution problems at street corners and junctions where the highest concentrations from traffic emissions arise and where human exposure is potentially greatest. This study is located in the Marylebone Road, Westminster and combines expertise from five universities. It will provide much needed measurements and lead to improvements in the modelling tools used to assess the impact of pollutants on health, including the accidental or terrorist releases of toxic or flammable gases, spatial design and the location of buildings. Other funded projects are studying the impact of pollution on plants and birds in the city and a major health study is currently in preparation.

APRIL host seminars and conferences, specialist workshops and meetings, and members participate at a national and international level at air quality events. The Network, established in 1999, is led by a steering committee chaired by Professor Helen ApSimon of Imperial College, London, supported by: Professor Mike Batty, UCL, Professor Bernard Fisher (Environment Agency), Professor Frank Kelly (King’s College), Professor Alan Robins (Surrey), Professor Lord Julian Hunt (UCL), Dr. Roy Colvile (Imperial), Dr. Steve Smith (King’s), Dr. Claire Burton (EPSRC), Dr. Janet Dixon (DEFRA), Jim Storey (Environment Agency (EA), David Hutchinson (GLA), Chris Lee (ALG), Steve Hedley (King’s College), Linda Davies (Network Co-ordinator, Imperial College). http://www.airpollution.org.uk [email protected]

Prof. Helen ApSimon: 0207 594 9292 Linda Davies: 0207 594 9295/07771 656509

Background and Executive Summary

The increase of oxidised nitrogen in urban areas and reduced nitrogen in rural areas has been widely recognised in Europe. This pilot study investigates the impact of reduced and oxidised nitrogen on sensitive vegetation at selected sites in England. The Review and Assessment of Air Quality (DETR, 2000) resulted in Air Quality Management Areas (DEFRA, 2000) being declared by twenty-nine of the thirty-three local authorities in London making it the largest urban area in the UK in breach of the Health Objectives for nitrogen dioxide of 21 ppb (annual mean). Objectives for total oxides of nitrogen (16 ppb) and sulphur dioxide (8 ppb) have been determined to protect sensitive vegetation and ecosystems from harm and although not applicable in urban environments, nevertheless are relevant. Both sites of conservation interest (e.g. SSSIs, cSACs and NNRs) and priority Biodiversity

Action Plan (BAP) species require special protection from nutrient (especially NH3) nitrogen deposition and it is therefore essential to develop sensitive biomonitoring methods appropriate for such habitats and species groups.

Data collected by Clapp (Clapp & Jenkin 2001*) of measurements of total NOx at the roadside in the centre of Westminster averaged 210 ppb NOx, falling to 73 and 23 ppb respectively at the outer London background sites of Hillingdon and Teddington. There are huge diurnal and seasonal variations in values. The modelling of nitrogen dioxide concentrations by the Greater London Authority (GLA, 2001) reproduced in Section 1, Figure 1.1 clearly demonstrates the gradient in nitrogen dioxide concentrations across London and was used to select three inner London sites and three outer London sites where the diversity, frequency and vitality of lichens and bryophytes were recorded on a single phorophyte, ash (Fraxinus excelsior). Lichen diversity on oak (Quercus spp) at one central location was also recorded. Background concentrations of nitrogen dioxide decline along a transect from central London through the suburbs and into Surrey where only three local authorities needed to declare Air Quality Management Areas. Heathland communities are particularly sensitive to nitrogen input and formed the basis of a second study looking at nitrogen accumulation in Calluna vulgaris and the use of isotopic signatures as an index of traffic-derived pollution in this species.

The primary emission sources of nitrogen in London are transport and heating, contrasting sharply with rural areas where reduced nitrogen from farming activities is frequently the most common form of atmospheric pollution. Intensive agricultural activity in the form of a poultry unit in Norfolk and a dairy farm in Devon provide the third study area where the influence of ammonia on bark pH and lichen communities as determined by van Herk (1999, 2001) was assessed.

Protocols using lichens have been developed to monitor ammonia pollution in The Netherlands that have defined ‘nitrophyte’ and ‘acidophyte’ indicator species (van Herk) as well as an EU recording method to detect environmental changes. Part of this pilot study tests the application of these protocols in the UK in selected urban and rural sites. More detailed investigations at the microscale level were carried out using lichens transplanted from a rural site to the centre of London at the Natural History Museum wildlife garden (Cromwell Road) to assess eco-physiological changes in selected species. In addition, the impact of urban NOx emissions on higher plants was studied, along a transect of decreasing concentrations away from the city centre. The following methods were tested:-

• The EU Directional Quantitative Lichen Monitoring protocol was used to detect changes in epiphytic lichen diversity on trunks of ash trees between sites in inner and outer London. • Twigs with a healthy lichen community were transplanted within a clean air site in Somerset and to the wildlife garden at the Natural History Museum adjacent to Cromwell Rd in the vicinity of NOx monitoring gauges. The health of individual thalli of selected species was assessed using chlorophyll fluorescence. • A transect study was carried out during winter 2001/2, to assess changes in shoot chemistry of

Calluna (heather) along a gradient of decreasing NOx pollution from central London to rural Surrey. Shoots were analysed for concentrations of nitrogen, carbon, phosphorus, lead, zinc and stable isotopes of nitrogen. • The van Herk method was tested on oak trees in rural localities in different climatic regions in SW (North Wyke, Devon) and E England (Thetford) in the vicinity of ammonia monitoring networks and Sites of Special Scientific Interest.

The pilot survey in London showed differences in lichen diversity on ash trees between inner and outer London sites correlating with concentrations of transport emissions. Foliar nitrogen concentrations in Calluna were positively related to NO2 concentrations and also decreased significantly with increasing distance from central London. Carbon:nitrogen ratios showed the opposite relationship with both NO2 and distance, with linear regressions again statistically significant. High phosphorus concentrations in central London were responsible for a significant increase in foliar N:P ratios with distance from the city centre and suggested that phosphorus availability would not limit plant response to nitrogen at any of the study sites. Unlike studies elsewhere using mosses, foliar lead and zinc concentrations do not appear to be a useful index of exposure to traffic-derived pollution in Calluna. This may be because the sites used in this study were typically located at distances of more than 150 m from major roads, or because higher plants are more effective at excluding heavy 15 metals than mosses or lichens. Delta N measurements in Calluna were positively related to NO2 concentrations and declined significantly with increasing distance from central London and increasing distance from major roads. This may suggest a decreasing contribution of traffic-derived NOx to the nitrogen nutrition of higher plants away from busy roads and in less urban areas, although nitrogen transformations within the soil (and plant) make it difficult to interpret this signal without further investigation.

The high diversity of lichens on oak trees in central London suggests that species recovery is not as limited by slow dispersal mechanisms as previously considered. Over a 3-month period transplanted material from Somerset to a roadside site across a NOx gradient in London showed great variation in persistence and vitality between species. The chlorophyll fluorescence data requires further testing and evaluation over a longer time period to develop its application as a biomarker of stress for NOx.

Foliar nitrogen concentrations in Calluna and nutrient ratios suggest that many sites in urban areas experience high deposition inputs of nitrogen and as such are at risk from changes in species interactions and ecosystem function. The widespread occurrence of ‘nitrophyte lichens’ (as defined by van Herk) in urban and rural sites investigated in the pilot project suggests that both oxidised and reduced nitrogen is readily assimilated by these lichens. Further work is required to establish the sensitivity of urban vegetation to realistic levels of both NO and NO2, to determine the implications of

* Daily daylight-averaged NOx, annual mean concentration using 1998-1999 data ppbv. 4 current and future emissions of traffic-derived pollution on urban ecology and to define indicators appropriate for London. It is to be expected that species new to Britain or science will be discovered as was found during surveys in the Netherlands.

Indices of nitrophyte and acidophyte species were correlated with ammonia levels (Thetford) and distance from source (North Wyke). However, many nitrophyte species defined in Holland were absent from the sites in the UK, suggesting that indicator species must be evaluated on a regional basis. The importance of both climate and pollution history in the UK was also highlighted by the survey. Highly pollution sensitive Lobarion species that are also indicators of ecological continuity were present at North Wyke whilst at Thetford acidophytes on ancient trunks in conservation sites testified to former acidification. In both sites younger trees and particularly twigs supported the majority of nitrophyte species.

NO2 and NH3 data from the nearest National Diffusion Tube / Ammonia Monitoring Network sites may not provide the most accurate representation of vegetation exposure to NOx / NH3 in urban green spaces and the wider countryside. This pilot study has highlighted the need for standardised biomonitoring recording techniques in combination with physico-chemical and modelling data. This is necessary to develop practical indicator scales appropriate to the UK that can be used to monitor the new pollution climate. The data from this pilot study offer an insight into temporal and spatial differences in selected sensitive species under changing environmental conditions. The project does not consider other primary or secondary pollutants from these sources, climate change or differences in management practice.

1. The Air Quality Strategy for England, Scotland, Wales and Northern Ireland (2000). Department of Environment, Transport and the Region. HMSO, London. 2. The Air Quality Regulations (2000). Department for the Environment, Food and Rural Affairs.

3. Clapp, L.J. & Jenkin, M.E. (2001). Analysis of the relationship between ambient levels of O3, NO2 and NO as a function of NOx in the UK. Atmospheric Environment 35: 6391-6405. 4. The Mayor’s Air Quality Strategy. A consultation document. (2001). Modelled Concentrations of Nitrogen Dioxide, 1999. Greater London Authority (GLA), Romney House, Marsham Street, London. 5. Van Herk, C.M. (1999). Mapping of ammonia pollution with epiphytic lichens in the Netherlands. Lichenologist 31: 9-20. 6. Van Herk, C.M. (2001) Bark pH and susceptibility to toxic air pollutants as independent causes of changes in epiphytic lichen composition in space and time. Lichenologist 33: 419-441.

This report has been compiled in three sections, corresponding to the separate projects funded under contract to DEFRA

Section 1. (two parts) Impacts of NOx pollution on lichens

Part 1. Corticolous Lichens in London

L. Davies, Imperial College, London P.W.James, Natural History Museum J.H. Chimonides, Natural History Museum O.W. Purvis, Natural History Museum

Part 2. Investigating the impact of NOx on transplants

Feliciano Cirimele, The Natural History Museum Sarah Honour, Imperial College, London Randolph Kricke, University of Essen, Germany Pat Wolseley, The Natural History Museum O. William Purvis, The Natural History Museum

Section 2. Impacts of urban NOx pollution on vegetation in London

Sally Power, Imperial College, Ascot Tilly Collins, Imperial College, Ascot

Section 3. Assessing the role of biological monitoring using lichens to map excessive ammonia (NH3) deposition in the UK.

Pat Wolseley, The Natural History Museum Peter James, The Natural History Museum

SECTION 1

Impacts of NOx Pollution on Lichens

PART 1: CORTICOLOUS LICHENS IN LONDON

1. Introduction 9 2. Site Descriptions 10 3. Methods 13 4. Results 15 5. Discussion 17 6. Conclusions 20 7. Recommendations 21 8. References 28

Appendices Appendix A Table 1 Species Numbers 22 Table 2 Species Frequency 23 Table 3 Girth & pH measurements 24

Appendix B Modelled concentrations of Nitrogen Dioxide in London (1999) (GLA). 25

Appendix C Figure 1 Ash Diversity by Site, Dendrogram 26 Figure 2 Ash Frequency by Tree, Dendrogram 26 Figure 3 Similarity below 50 cm (Dog Zone), Dendrogram 27 Figure 4 Ash pH ranges 27

Authors Linda Davies, Imperial College, London Peter W. James, Natural History Museum Jim H. Chimonides, Natural History Museum O.William Purvis, Natural History Museum

Acknowledgements The authors would like to acknowledge the assistance of: Greater London Authority Natural History Museum: Simone Louwhoff Amanda Waterfield Len Ellis Dave John Dr. I. Blatchley Prof. H.J. Beker

1. INTRODUCTION

This pilot study investigates the diversity and distribution of corticolous lichens on a single phorophyte, ash (Fraxinus excelsior) at six sites in London and on oak (Quercus) at one site. It was proposed as part of a wider study covering all thirty-two London Boroughs and the City of London recording corticolous lichens, bryophytes and fungi on five Fraxinus at each site and young oak (Quercus) at selected sites.

The project investigates both spatial and temporal trends in relation to species diversity, vitality and community structure, making reference to the EU Limit values (Air quality framework Directive, 1996) to protect sensitive species from the effects of sulphur dioxide and oxides of nitrogen. The annual mean of 16 ppb (30 µg/m3) determined for oxides of nitrogen (NOx) is widely exceeded in London. In contrast the sulphur dioxide annual mean and winter mean (1 Oct to 31 Mar) of 8 ppb (20 µg/m3) are rarely exceeded. These values have been incorporated into the Air Quality (England) Regulations 2000 (DETR, 2000). They do not apply in urban environments but nevertheless are critical levels for the protection of sensitive species. Lichens and bryophytes are less well protected from atmospheric pollutants than higher plants because they do not possess cuticles or stomata. Some species, however, are better adapted and demonstrate greater tolerance than others. Only one corticolous species, Lecanora dispersa was recorded in central London in 1967 (Laundon, 1970) at a time when the limited diversity in most urban environments was attributed to the impact of sulphur dioxide.

This study applies, for the first time in the UK, the proposed EU Quantitative Lichen Monitoring protocol proposed as a standardised recording technique to assess lichen diversity as an indicator of environmental change.

For this pilot study Westminster, Southwark and Tower Hamlets were selected as the inner London sites, all within 6 km of Charing Cross, and outer London locations were selected in Enfield, Harrow and Bromley. A contour map prepared by the Greater London Authority demonstrating the concentration gradient of nitrogen dioxide decreasing with distance from central London to the suburbs is provided (Appendix B).

2. SITE DESCRIPTIONS

2.1 Inner London 2.1.1 Westminster Regent’s Park (TQ 282 833) Distance from Charing Cross 3 km

Regent’s Park evolved from a 16th Century Royal Hunting Estate into a public park in the early 1800s. Huge areas of trees had been removed to meet timber demands, creating a major challenge in landscaping and design on an area overlying a great depth of London clay. An ambitious tree planting programme, formal gardens and ornamental lakes have transformed the park into a major recreational centre with a wide variety of flora and fauna covering a total area of over 170 hectares.

Ash trees in the vicinity of the York Bridge entrance on the Outer Ring Road, close to the Marylebone Road were selected for the survey, and young oaks adjacent to the Heather Garden. The oaks are situated further from the Inner Road, away from traffic influences and are sheltered by a children’s play area. They are closer to London Zoo, and adjacent to the Bird House and lake.

Air pollution data from the kerbside Marylebone monitoring station 0.5 km from York Bridge is provided below but background estimated concentrations calculated by Westminster Local Authority using 1997 data suggest that lowest concentrations of nitrogen dioxide at the present time would be in the region of 31 ppb. The range of total NOx measured at this site is between 50 and 500 ppb (Clapp & Jenkin, 2001).

Annual Average NO2 1997 Background 31 ppb

Annual Average NO2 1999 Kerbside 47 ppb

Annual hourly max NO2 1999 Kerbside 169 ppb

Annual Average SO2 1999 Kerbside 5 ppb

Nitrogen dioxide measurements for all sites are taken from the SEIPH 1999 Annual Report (SEIPH, 2001), unless otherwise stated, and represent measurements recorded by the London Air Quality Network.

2.1.2 Southwark Burgess Park (TQ 333 778) Distance from Charing Cross 4 km

This recreational Park was created in the post war period for the enjoyment of the people of Southwark and includes several ornamental lakes, play areas and landscape features with a wide variety of trees, including lime, plane, and ash, many shrubs and cultivated beds. It 10 covers an area of approximately thirty-five hectares and lies between two major road links. Background concentrations of nitrogen dioxide measured in 1997 with diffusion tubes give an average annual value of 21 ppb in the centre of the park (Southwark, 2001):

Annual Average NO2 1999 Roadside 39 ppb

Annual hourly Max NO2 1999 Roadside 112 ppb

Annual Average SO2 1999 Background 4 ppb

SO2 values are taken from the DEFRA website maintained by AEA/NETCEN (AEA, 2001).

2.1.3 Tower Hamlets Victoria Park (TQ 353 835) Distance from Charing Cross 6 km

This park was created in 1888 in response to the demands of local residents for recreational space in East London. It extends across some 35 hectares and is beautifully landscaped with a variety of mixed broadleaf trees, shrubs, ornamental beds and two lakes. The park is bordered on both sides by major transport links and modelled data (Tower Hamlets, 2002) suggest that background concentrations of nitrogen dioxide remain at approximately 26 ppb in the surveyed areas.

Annual Average NO2 2002 Background 26 ppb

Annual Average NO2 1999 Roadside 34 ppb

Annual hourly Max NO2 1999 Roadside 119 ppb

Annual Average SO2 1999 Roadside 3 ppb

2.2 Outer London 2.2.1 Bromley Jubilee Country Park (TQ 436 680) Distance from Charing Cross 18 km

This country park is a secondary woodland with remnants of ancient woodland, pasture and cultivated areas covering approximately 25 hectares. It has many small mixed areas of oaks, ash and willow supported by open glades with old field boundaries, hedges and ponds enhancing the open aspect. The western end of the park is dominated by hawthorn with elder, pine and birch. Exceedences of the nitrogen dioxide Objectives are not expected in Bromley, unlike most other London boroughs, with background levels modelled at 15ppb.

Additional modelled data on NO2 by the local authority provides the following information:

NO2 Annual Average Roadside 16 ppb

NO2 Hourly Maximum 55 ppb

SO2 Annual Average 3 ppb 11

2.2.2 Enfield Covert Way Nature Reserve (TQ 263 974) Distance from Charing Cross 17 km

This secondary woodland covering approximately 10 hectares, is set within a quiet sheltered suburban location close to Hadley Common. It is rich in mature oak and ash which are quite densely planted in parts, with a small area of grassland in the centre. A return to coppicing within the site has created a more open aspect complementing the denser plantings in this small conservation area, maintaining a good bryophyte flora and a haven for avian populations.

1999 NO2 Annual Mean Background 18 ppb

1999 NO2 Hourly Background Max 90 ppb

1999 NO2 Annual Mean Roadside 24 ppb

1999 NO2 Hourly Roadside Max 93 ppb

1999 SO2 Annual Mean 3 ppb

2.2.3 Harrow Canons Park, Stanmore (TQ 183 915) Distance from Charing Cross 16 km

This is an 18th Century Estate with some formal features retained, but now managed as open parkland comprising a narrow mixed broadleaf woodland area, open aspect trees, including both ancient and recently planted oaks and a spinney of younger oak, ash, sycamore and elm. Some planted garden areas and ornamental features remain.

1999 NO2 Annual Mean Suburban 18 ppb

1999 NO2 Hourly Max Suburban 86 ppb

1999 SO2 Annual Average 3 ppb

3. METHODS

3.1 Tree selection Ash was selected as the major phorophyte as it is widely distributed across London, with oak as the second species. Due to time and budget constraints only six sites were selected and surveys of ash carried out at all sites with oak being examined at Regent’s park only. Eight bark pH measurements were taken per tree. Trees were selected to meet the following criteria: unbranched below 200 cm, upright, open aspect, without injury or disease with a minimum girth of 50 cm and a maximum girth of 150 cm. They were selected at a minimum distance of 150 metres from the park entrance in order to reduce roadside influence. The first five trees meeting the selection criteria were surveyed.

3.2 Lichen Sampling Strategy The approach follows that of Asta et al. (2002). A narrow five-laddered quadrat was attached to the tree at a height of 150-cm using stainless steel pins at each intersection. The quadrat comprises five grid squares, each measuring 10 x 10 cm extending to cover 50 cm in depth. It was positioned at four orientations, North, South, East and West. All lichen species in each quadrat were recorded and given a value of 1, allowing a maximum frequency score of 5 per quadrat and 20 per tree for each species. The Lichen Diversity Value (LDV) for each site is then calculated by adding the frequencies of all species in each quadrat segment and dividing by the number of trees examined (5). In addition, all species below 50 cm and all species above 50 cm up to 200 cm were recorded and any special features noted.

3.3 Bark Acidity Measurements Bark pH readings at positions 1 and 5 in the quadrat were measured using a flathead electrode (HI 8014 pH meter and BDH Gelplas double junction Flat Tip electrode 309/100/09) according to Looney & James (1988) and Farmer et al. (1990). The bark was moistened with a solution of KCl (0.1 mol) and the reading taken after three minutes, immediately following a second application of KCl.

3.4 Bryophytes, Algae, Fungi and Liverworts All epiphytes were recorded

3.5 Species identification Three techniques were employed for identification: Field examination using a x10 hand lens and chemical tests (Orange, 2002) Microscopical examination of thalli and spores Thin Layer Chromatography (Orange, 2002)

The nomenclature follows lists maintained by Brian Coppins (British Lichen Society) (see http://www.argonet.co.uk/users/jmgray/syn.htm) 13

3.6 Statistical methods Correlations were calculated using Excel. Species composition and frequency was investigated using multivariate ordination analysis that serves to reduce complex species- site data to a form that is visually interpretable. While all multivariate analyses were based on the raw data, additional analyses were performed on transformed (double square root; presence-absence) to detect any improved relationships when effects of dominance were reduced. The methods used were:

Non-Metric Multidimensional Scaling (MDS): data from the 10 stations were subjected to analysis using MDS that is currently widely used in the analysis of spatial and temporal change (e.g. Warwick & Clarke, 1991). The recorded observations from the 6 stations were exposed to computation of triangular matrices of similarities between all pairs of samples. The similarity of every pair of sites was computed using the Bray-Curtis index on the raw and transformed data. Clustering was by a hierarchical agglomerative method using group average sorting, and the results are presented as a dendrogram and as a two-dimensional ordination plot.

SIMPER: the MDS clustering program was used to analyze differences between sites. SIMPER (Plymouth Marine Laboratories PRIMER package) enables those species responsible for differences to be identified by examining the contribution of individual species to the similarity measure.

CONPLOT: this program (Plymouth Marine Laboratories PRIMER package) permits environmental variables to be superimposed on MDS plots, giving a visual indication of correlation between clusters and environmental factors (i.e. pH).

4. RESULTS

4.1. Lichens Species Diversity

A total of 56 different species were recorded. The data are presented in Table 1. (Appendix A)

• Highest diversity was recorded on ash at the two outer London sites of Harrow and Enfield, with 35 species at each site. The third outer London site, Bromley yielded 29 species.

• The three inner London sites all carried 20 species.

• 32 species were recorded on oak in Regents Park.

Fraxinus : 24 species Arthonia spadicea, Bacidia arceutina, B. laurocerasi, B. naegelii, Caloplaca phlogina, Candelariella reflexa, C. vitellina, Cliostomum griffithii, Dimerella pineti, Diploicia canescens, Hyperphyscia adglutinata, Hypotrachyna revoluta, Lecanora dispersa, L. muralis, Lecanora saligna, Micarea prasina, Phlyctis argena, Physcia aipolia, P. caesia, P. dubia, Punctelia ulophylla, Rinodina subexigua, R. exigua and Strangospora pinicola.

Quercus: 6 species Lecanora albella, Parmelina tiliacea, P. saxatilis, Physconia grisea, Pleurosticta acetabulum, Rinodina gennarii.

Common to both tree species: 26 species Amandinea punctata, Bacidia delicata, Candelaria concolor, Evernia prunastri, Flavoparmelia caperata, Flavoparmelia soredians, Hypogymnia physodes, Lecanora carpinea, L. chlarotera, L. conizaeoides, L. expallens, L. symmicta, Lecidella elaeochroma, Lepraria incana, Melanelia subaurifera, Parmelia sulcata, Parmotrema chinense, Phaeophyscia orbicularis, Physcia adscendens, P. tenella, Punctelia subrudecta, Ramalina farinacea, Scoliciosporum chlorococcum, Xanthoria candelaria, X. parientina and X. polycarpa.

Species common to all sites: Amandinea punctata, Bacidia delicata, Parmelia sulcata, Physcia adscendens and P. tenella.

4.2 Lichen Diversity Values (LDV’s) Highest values were recorded at Harrow, followed by Southwark, and Bromley (Appendix A, Table 2):

4.3. Other corticolous flora on Fraxinus excelsior 12 Bryophytes: Amblystegium serpens, Brachythecium rutabulum, Dicranoweisia cirrata, Dicranum scoparium, Eurhynchium praelongum,Grimmia pulvinata, Hypnum cupressiforme, H.andoi, Orthotrichum affine, O.diaphanum, O.lyellii, Ulota crispa. 2 Parasitic Fungi: Lachnella alboviolascens, Gloniopsis praelonga 2 Liverworts: Lophocolea bidentata, Frullania sp. 4 Algae: Desmococcus viridis, Athelia arachnoidea, Prasiola crispa, Trentepohlia sp.

4.4 Statistical Analysis 4.4.1 Species Numbers Dendrograms based on species presence on ash at each site (Appendix C, Figure 1) and ash and oak combined demonstrate that similarity percentage contributions lead to a division at the sixty percent level into inner and outer London sites for ash. Flora on the oaks in Regent’s Park are more closely aligned to the Outer London sites.

4.4.2. Species Frequency In all cases quadrat segments (NSWE) cluster together in the 3 principal groups, apart from the north segment quadrat at Tower Hamlets. Bromley trees initially separate at the eighteen percent level (i.e. these are the most distinct) with two further major groups: the first comprising, Southwark, Regents and Tower Hamlets (apart from the north segment) with Enfield, Harrow and the north segment of Tower Hamlets closely aligned (Appendix C, Figure 2).

4.4.3 Species below 50 cm The dendrogram (Appendix C, Figure 3) suggests a difference between those sites with more dense housing located away from residential areas and those of a more densely populated location where the dog zone would be most influential in creating an eutrophicated substrate in addition to nitrification from anthropogenic emissions. Xanthoria parientina and X. polycarpa were not recorded in Bromley although they were widespread and frequent at the other sites and are typical species of nitrogen enriched environments. Further details on the origins of the designation ‘nitrophyte’ and ‘acidophyte’ can be found in the Section 2: Assessing the role of biological monitoring using lichens to map excessive ammonia (NH3) deposition in the UK, Appendix 1.

4.5 Girth and pH. The girth and pH measurements for the sites are given in Table 3 (Appendix A). Ranges on ash were from 3.7 to 5.59 and 2.7 to 5.19 on Oak. The lowest Ash pH was recorded in Bromley. The data presented in Figure 4.5 illustrate that Ash trees at Bromley have a significantly lower bark pH as determined by a Spearman rank correlation coefficient (p = 0.01).

4.6 Vitality Few signs of poor health were recorded except at Canons Park, Stanmore, where some of the macrolichens were dying. However, the largest thalli of Parmelia sulcata and Flavoparmelia caperata were measured at this site. Thalli of Hypogymnia physodes, Evernia prunastri and Ramalina farinacea were generally below normal at less than 0.5 cm. Thalli of Physcia species at the Enfield site were numerous, but in the region of 0.5 cm, possibly just colonising the recently coppiced woodland area compared with the very well developed populations at the inner London sites.

5. DISCUSSION

This pilot study investigated the distribution of corticolous lichens at six sites in London. As so few sites have been studied results should therefore be interpreted with care. However there are some interesting results that require further consideration. The species number is very high for such a small sample of only thirty-five trees and the species recorded include many not seen for over fifty years, as well as some never recorded in London before (James et al. 2002). In particular, the macrolichen Pleurosticta acetabulum and the crustose Strangospora pinicola represent a flora uncommon to urban environments in recent decades.

5.1 Temporal and Spatial Trends Species numbers have increased significantly over the past fifty years and correlate with falling concentrations of sulphur dioxide (all below the critical level of 8 ppb) and increasing concentrations of nitrogen and other transport emitted pollutants as demonstrated in Figs 1 and 2.

Fig. 1 Fig. 2

Lichen species numbers vs NO2 concentrations (ppb) Lichen Diversity Value and NO2 concentrations with increasing distance from Charing Cross with increasing distance from Charing Cross

40 35 70.0 35 35 30 60.0 30 30 25 50.0 25 25 20 40.0 20 20 15 LDV 30.0 15

15 NO2 (ppb) 20.0 10 (ppb) NO2 10 10 10.0 5 5 5 0.0 0 Lichen species nos (5 ash trees) 0 0 r ts y d e rk e e l k ld st a fie lets row ley ie w ml ml f in th a Harrow En Har En m u H Bro Ham Brom st r stminster e So e e Southwar w W W o Tower T

Laundon (1970) analysed species diversity and distribution in London in the late 1960s. He observed the decline in urban populations to a single species in central London, Lecanora dispersa, and a total of nine corticolous species were recorded throughout London (an area defined within a radius of 16 km from Charing Cross).

Significant increases in populations were recorded in 1980 (Rose & Hawksworth 1981) and 1988 (Hawksworth & McManus, 1988) with total corticolous and lignicolous species on mixed tree species across fifty sites recorded as 49, 17 of which were found in Regent’s Park. Both these and more recent surveys (Bates et al 1990, 1992, 2001) have referred to the slow reinvasion rates for specific species in London, and noted a change in community structure with the widely distributed species Hypogymnia physodes and Parmelia saxatilis decreasing and an increase in Flavoparmelia caperata and Physcia aipolia.

Spatial distribution suggests that Numbers of lichen species on 5 ash trees vs distance inner London ash carry a from Charing Cross

y = 0.9447x + 16.256 significantly lower species range 40 R2 = 0.8076 than ash in outer London areas 35 30 (Fig. 3). The use of a single 25 phorophyte for this study has species of No 20 15 identified a trend not obvious from 0.00 5.00 10.00 15.00 20.00 other recent studies when lichen Distance from Charing Cross (Km) diversity on mixed tree species and Fig. 3 other substrates are recorded. The young oaks were in a very sheltered position but

18 nevertheless suggest that dispersal mechanisms are bringing a rich diversity of species into the centre of London.

The inner London sites carry many of the characteristics of the Xanthorion (James, 1974) community and include a high percentage of species favouring high nitrogen environments. The communities recorded on the Oaks resemble more closely those of the Parmelia caperata –Pertusaria spp. community described as characteristic of open woodland: Lecanora chlarotera, Flavoparmelia caperata, Parmotrema chinense, Parmelia saxatilis and P. sulcata.

5.2 Bark pH and Girth Lichens are very sensitive to bark pH and many species are limited to specific ranges. Using two phorophytes therefore increases the range of potential diversity. It is interesting to note the lower bark pH of the ash in Bromley where transport emissions and dog zone influences were at their lowest within the study area

The pH and girth of the oaks demonstrate their young age and may account for the large number of species recorded, contrasting sharply with the small number of species on mature oaks in Regent’s Park (communicated, Davies L.) and Kensington Gardens (Bates et al 1990, 2001).

5.3 Air Quality Annual average concentrations of sulphur dioxide across the area are within the recommended Objectives for sensitive vegetation. Nitrogen dioxide concentrations in Inner London range from 50 at roadside locations and 31 at background locations to a low of 14 ppb at outer London sites. Data for nitric oxide are not included in the study but concentrations in London would add significantly to the total NOx concentration as highlighted by Clapp & Jenkin (2001).

6. CONCLUSIONS

The large number of species recorded in this pilot survey demonstrates a significant increase in lichen diversity in London in recent decades.

However, a comparison of species on ash suggests a major difference in species number and composition between the inner and outer London sites. We have identified a trend towards increasing diversity with distance from Charing Cross. The importance of using the same substrate for comparative purposes is highlighted and further enhanced by the diversity on young oaks in Regents Park (James et al. 2002). This study utilises for the first time the proposed EU Quantitative Lichen monitoring methodology. The approach provides a standardised recording technique to assess lichen diversity as an indicator of environmental change.

Both nitric oxide and nitrogen dioxide are toxic to sensitive species, but very few studies have investigated this effect. Concentrations of NO are higher in central London and frequently exceed nitrogen dioxide concentrations.

The high number of ‘nitrophytes’ suggests that oxidised nitrogen is readily assimilated by many lichen species and is encouraging species considered indicative of eutrophicated areas, particularly on the urban fringe. These results are related to the data collected in other parts of this study.

The disappearance of some species preferring a more acidic substrate was noted during this study.

The survey suggests that species diversity in London is perhaps not as limited by slow dispersal mechanisms as previously considered, although development may be arrested in sensitive species as demonstrated by the frequency of small thalli of several species such as Evernia prunastri and Ramalina farinacea and their rarity at other sites The presence of fertile species reproducing by sexual reproduction only (designated F, in Table 1, Appendix A) is interesting and requires further evaluation. An intriguing possibility is that some lichens in London may have existed in forms unrecognisable to lichenologists waiting until conditions are appropriate for development of typical thalli, a phenomenon previously suggested for the lichen Xanthoria parietina by Ott (1987). Most lichens identified, however, are able to reproduce asexually using isidia (I) or soredia (S), detachable outgrowths on the thallus containing the photobiont.

7. RECOMMENDATIONS

• The study area should be extended to cover more sites in London to investigate further the association between lichen diversity and oxides of nitrogen identified by this pilot study. In particular the study should include roadside locations where the impact of NO will be highest, contrasting with the background locations selected in this study.

• Species demonstrating a limited tolerance range should be selected for study at a physiological level.

• This study suggests that sensitive species may respond differently to oxidised nitrogen across a range of concentrations and in different forms. These aspects require further investigation particularly in relation to the Objectives for Sensitive Vegetation and Ecosystems. London provides an ideal laboratory to study the effects of oxidised nitrogen. Ambient concentrations of nitrogen dioxide are the highest in the UK (GLA 2001), nitric oxide is particularly high in inner London and ozone and ammonia generally lower than in rural environments.

• The results of this part of the study support the need for a joint project to investigate further the uptake and effect of oxidised and reduced nitrogen on sensitive plant species in relation to current critical levels for nitrogen dioxide and the impact of ammonia.

• The EU draft protocol for lichen biomonitoring used in this report, whilst offering a valuable protocol for quantifying lichen diversity, requires further evaluation concerning assessment of nitrogen emissions.

• The information gained through the above studies should be used as a precursor to developing practical indicator scales and in the development of their regulatory role to complement physicochemical monitoring and modelling studies.

APPENDIX A Table 1 Species Diversity Summary. N= nitrophytes; A = acidophytes; F = fertile; S = sorediate; I= isidiate Summary Regents South- Tower Enfield Harrow Bromley Total Regents Total N & A F & S park wark Hamlets park Fx Fx Fx Fx Fx Fx Fx All Amandinea punctata 1 1 1 1 1 1 6 1 7 F Arthonia spadicea 1 1 1 F Bacidia arceutina 1 1 2 2 S Bacidia delicata 1 1 1 1 1 1 6 1 7 S Bacidia laurocerasi 1 1 1 F Bacidia naegelii 1 1 1 F Caloplaca phlogina 1 1 1 S Candelaria concolor 1 1 1 2 S Candelariella reflexa 1 1 1 1 1 5 5 N S Candelariella vitellina 1 1 1 1 1 5 5 N F Cliostomum griffithii 1 1 2 2 F Dimerella pinetii 1 1 2 2 F Diploicia canescens 1 1 1 S Evernia prunastri 1 1 1 1 1 5 1 6 A S Flavoparmelia caperata 1 1 1 1 4 1 5 S Flavoparmelia soredians 1 1 1 2 S Hyperphyscia adglutinata 1 1 1 3 3 S Hypogymnia physodes 1 1 2 1 3 A S Hypotrachyna revoluta 1 1 2 2 F Lecanora albella 0 1 1 F Lecanora carpinea 1 1 2 1 3 F Lecanora chlarotera 1 1 1 3 1 4 F Lecanora conizaeoides 1 1 1 3 1 4 A SF Lecanora dispersa 1 1 1 1 1 1 6 6 N F Lecanora expallens 1 1 1 1 4 1 5 S Lecanora muralis 1 1 1 N F Lecanora saligna 1 1 1 F Lecanora symmicta 1 1 2 1 3 F Lecidella elaeochroma 1 1 1 1 4 1 5 F Lepraria incana/lob 1 1 1 1 4 1 5 A S Melanelia subaurifera 1 1 1 1 1 5 1 6 S Micarea prasina 1 1 1 SF Parmelia saxatilis 0 1 1 A I Parmelia sulcata 1 1 1 1 1 1 6 1 7 S Parmelina tiliacea 0 1 1 I Parmotrema chinese 1 1 1 3 1 4 S Phaeophyscia orbicularis 1 1 1 1 1 5 1 6 N S Phlyctis argena 1 1 1 S Physcia adscendens 1 1 1 1 1 4 1 5 N S Physcia aipolia 1 1 1 3 3 F Physcia dubia 1 1 2 2 N S Physcia sp.* 1 1 1 1 1 1 6 7 S Physcia tenella 1 1 1 1 1 1 6 1 7 N S Physconia grisea 0 1 1 S Physica caesia 1 1 1 N S Pleurosticta acetabulum 0 1 1 F Punctelia ulophylla 1 1 1 S Punctelia subrudecta 1 1 1 1 4 1 5 S Ramalina farinacea 1 1 1 1 4 1 5 S Rinodina gennarii 0 1 1 N F Rinodina subexigua 1 1 1 F Rinodina exigua 1 1 2 2 F Scoliciosporum chlorococcum 1 1 1 1 1 5 1 6 SF Strangospora pinicola 1 1 1 3 3 F Xanthoria candelaria 1 1 1 1 1 1 5 1 6 N S Xanthoria parietina 1 1 1 1 1 5 1 6 N F Xanthoria polycarpa 1 1 1 1 1 5 1 6 N F 20 20 20 35 35 28 32

Table 2. Frequency Values: Lichen Diversity Values (Asta, 2002) North South East West Totals Westminster 16.8 9.6 6.3 11.0 43.6 Southwark 12.8 16.0 11.8 9.4 50.0 Tower Hamlets 8.6 11.2 8.4 10.8 39.0 Harrow 11.4 19.0 15.6 14.2 60.2 Bromley 4.5 22.8 8.8 12.8 48.9 Enfield 10.2 15.0 8.8 11.0 45.0

The Lichen Diversity Value is calculated by taking the sum of the frequency of each species as recorded in the total quadrat for the relevant orientation and dividing by the number of trees per site. The totals are then summed to give a final diversity score (lichen diversity value ‘LDV’) per site

Regents Tower Hamlets Southwark Enfield Harrow Bromley R1 R2 R3 R4 R5 T1 T2 T3 T4 T5 S1 S2 S3 S4 S5 E1 E2 E3 E4 E5 H1 H2 H3 H4 H5 B1 B2 B3 B4 B5 Ash Gir 96 77 107156 82 65 58 65 52 71 76 79 90 84 92 116 127 70 58 70 50 68 82 91 46 99 108 91 76 133 pH S1 4.5 5 5.1 4.4 4.4 5.5 5.6 4.8 4.7 5.4 4.3 4.5 5 5.3 4.7 4.9 5.6 4.9 5.1 4.95555.354.34.44.54.24.5 S5 5.2 4.9 5.3 5.2 4.6 5.5 5.2 5.1 4.9 5.4 4.95554.84.55.45.1 5 5.2 5 4.9 5 5.1 4.9 4.9 4.7 4.6 3.9 4.7 E1 4.7 4.7 5.2 4.7 4.7 5.6 4.9 5.2 5 5.1 4.9 4.5 5.1 5.3 5 5 5.8 5.1 5.1 5.1 5.1 5.1 5 5.3 4.8 3.6 4.3 4.1 3.8 4.9 E5 5 5 5.2 4.2 4.9 5.5 5.2 5.3 5.1 5.3 5 4.9 5.4 5.2 5.1 5.4 5.5 5.1 5 5 4.9 5.2 5.2 5.2 5 4.1 4.4 4.2 3.9 4.9 N1 4.8 4.9 5.2 4.3 4.9 5.8 5.4 5.1 5.1 4.9 4.6 4.5 5.2 5.6 5.3 5.5 5.5 5.2 5.1 5.5 4.9 5 5 5.2 4.2 3.6 4.2 3.7 4.2 3.9 N5 5 4.9 5.1 4.2 4.7 6 5.4 5.1 5.1 5.1 4.8 4.8 4.8 5.4 5.3 5.8 5.6 5.2 5.1 5.6 4.9 5.1 4.9 5 4 4.7 4.2 3.7 3.6 4.3 W1 4.8 5 4.9 4.4 4 5.5 5.5 5.1 5.3 5.5 4.4 4.7 5.1 4.7 5.2 5.3 5.6 5.3 5.3 4.9 5.1 5.2 5.4 5.9 4.7 3.9 4.3 4 3.8 4.6 W5 4.9 5.2 5.3 4.3 4.1 5.8 5.4 5.1 5.4 5.4 4.9 5.5 4.8 4.8 5.3 5.7 5.7 5.3 5.5 4.8 5 5.2 5.4 5.3 4.8 4.5 4.6 4.3 4 4.4 Oak Gir 51 51 62 98 53 pH S1 4.2 4.1 4.2 3.2 5 S5 4.2 4.3 4.2 3.8 4.9 E1 4 4.4 4.7 3.3 4.8 E5 4 4.4 4.7 3.9 5.2 N1 4.3 4.6 4.4 3.3 4.7 N5 4 4.2 4.3 2.7 5.1 W1 4.5 4.3 4.5 3.4 4.7 W5 4.5 4.1 4.5 3 4.7 pH readings - S1 indicates position 1 in quadrat (10x10 cm) at a height of 150 cm(top of quadrat), S5 indicates position 5 at base of quadrat at a height of 60 cm (top of quadrat facing South (then East, North, West) (top of quadrat facing South (then East, North,West)

Table 3. Girth and pH measurement

APPENDIX B

Figure 1.1 Modelled Concentrations of Nitrogen Dioxide in London (ppb) 1999 (1997 weather data) Source: Greater London Authority

ppb NO2

APPENDIX C

Figure 1. Species number on Ash by site (PRIMER): A = Outer London Sites. B = Inner London

70 Similarity

80 A B

E H BTR S 100

Figure 2. Ash Frequency Data (PRIMER)

Similarity 60

100 TS TE TN BS BE SE SS ES EE BN SN RE RS HS HE EN HN RN TW BW SW EW RW HW

Figure 3. Similarity below 50 cm (Dog Zone) (PRIMER)

Ash

60 Similarity

100 TT1< 50cm ST1< 50cm ST1< BT1< 50cm BT1< 50cm ET1< HT1< 50cm RT1< 50cm

Figure 4. Ash pH ranges (PRIMER)

Ash

Stress: 0.16

E3 R4 E2 E5 R1 R5 H5

R3 B1 E4 R2 T3 T2 B5 H1 B2 T1 T5 H4 H3 H2

B3 S4 S5 B4 S1 S3

8. REFERENCES

Air Quality Framework Directive (Council Directive 96/62/EC) See http://www.europa.eu.int/comm/environment/air/ambient.htm. AEA Technology/NETCEN (1998) Annual Report. Asta, J., Erhardt, W., Ferretii, M., Fornasier, F., Kirschbaum, U., Nimis, P.L., Pirintsos, S., Purvis, O.W., Scheidegger, C., Van Haluwyn, C. & Wirth, V. (2002). Mapping Lichen Diversity as an Indicator of Environmental Quality. In Nimis, P.L., Scheidegger, C. & Wolsely, P.A. (Eds.) Monitoring with Lichens. Kluwer, Dordrect: 273-279. Bates, J.W., Bell, J.N.B. & Farmer, A.M. (1990). Epiphyte recolonisation of oaks along a gradient of air pollution in South-east England, 1997-1990. Environmental Pollution 68: 81-99. Bates, J.W. & Farmer, M.A. (Eds.) (1992). Bryophytes and Lichens in a Changing Environment. Clarendon Press, Oxford. Bates, J.W., Bell, J.N.B. & Massara, A.C. (2001). Loss of Lecanora conizaeoides and other fluctuations of epiphytes on oak in S.E. England over 21 years with

declining SO2 concentrations. Atmospheric Environment 35: 557-568. Bromley Local Authority (2000). Review and Assessment of Air Quality in Bromley. Environmental Health Department, Bromley Council. Clapp, L.J. & Jenkin, M.E. (2001) Analysis of the relationship between ambient

levels of O3, NO2 and NO as a function of NOx in the UK. Atmospheric Environment 35: 6391-6405. DETR (2000) The Air Quality Strategy for England, Scotland, Wales and Northern Ireland. Working Together for Clean Air. Department of the Environment, Transport and the Regions, in partnership with the Scottish Executive, The National Assembly for Wales, and the Department of the Environment for Northern Ireland. Available on-line at http://www.environment.detr.gov.uk/airquality/index.htm Farmer, A.M., Bates, J.W. & Bell, J.N.B (1990). A comparison of methods for the measurement of bark pH. Lichenologist 22: 191-197. Hawksworth D.L., & McManus P. (1989). Lichen recolonisation in London under conditions of rapidly falling sulphur dioxide levels and the concept of zone skipping. Bot. J. Linn.Soc. 100: 99-109. James, P.W., Purvis, O.W. & Davies, L. (2002). Epiphytic Lichens in London. Bulletin of the British Lichen Society. 90: 1-3. Laundon, J.R. (1970). London’s Lichens. The London Naturalist: 49: 2-69. Looney, J.H. & James, P.W. (1988). Effects on lichens. In Acid Rain and Britain’s Natural Ecosystems (M.A. Ashmore, J.N.B. Bell & C. Garretty, eds): 13-25. London: Imperial College Centre for Environmental Technology Mayor’s Draft Air Quality Strategy (2001). Greater London Authority (GLA), Romney House, Marsham Street, London SW1. 28

Orange, A., James, P.W., White, F.J. (2002). Microchemical methods for the identification of Lichens, British Lichen Society Ott, S (1987) Reproductive strategies in lichens. - In: E. Peveling (ed.): Progress and Problems in Lichenology in the Eighties. Bibliotheca Lichenologica No. 25. J. Cramer, Berlin-Stuttgart, pp. 81-93. PRIMER, Plymouth Routine in Marine Environmental Research. Non-metric multidimensional scaling (Bray Curtis 1957). Rose, C.I. & Hawksworth, D.L. (1981). Lichen recolonisation in Cleaner Air. Nature 289: 289-292. Rose, F. & James, P.W. (1974). Regional Studies on the British Lichen Flora 1. The corticolous and lignicolous species of the New Forest, Hampshire. Lichenologist 6: 1-72 SEIPH (1999) Annual Report. Air Quality in London, SEIPH-ERG, King’s College, London. Southwark Local Authority, Diffusion Tube Survey 1997. Tower Hamlets (2002) Nitrogen dioxide modelled contour map. Warwick, R.M. & Clarke, K.R. (1991) A comparison of some methods for analysing changes in benthic community structure. J. mar. biol. Ass. U.K., 71: 225-244. World Health Organisation Air Quality Guidelines for Europe (2000). Published by WHO, Regional Office for Europe, Scherfigsvej 8, DK-21-00 Copenhagen, Denmark

PART 2: INVESTIGATING THE IMPACT OF NOx ON TRANSPLANTS

1. Introduction 31 2. Objectives 32 3. Materials and Methods 32 4. Results 35 5. Discussion 37 6. Conclusion 40 7. Recommendations 40 8. References 48

Appendices Appendix A: Estimated Air Pollution Data for the Wildlife Garden, 7 Nov 2001-15 March 2002 41 Appendix B Measurements used to estimate the NO and NO2 relationship for the 5 Sites in the Wildlife Garden 42 Photographs of Lichen Transplants Appendix C: Somerset (Transplant Site) 43 Appendix D: Parmelia sulcata. NHM Wildlife Garden (Site 2) 44 Appendix E: Xanthoria parietina NHM Wildlife Garden (Site 2) 45 Appendix F: Parmelia sulcata. NHM Wildlife Garden (Site 4) 46 Appendix G: Xanthoria parietina NHM Wildlife Garden (Site 4) 47

Authors Feliciano Cirimele, The Natural History Museum Sarah Honour, Imperial College, London Randolph Kricke, University of Essen, Germany Patricia Wolseley, The Natural History Museum O. William Purvis, The Natural History Museum

Acknowledgements The authors would like to acknowledge the assistance of: The Natural History Museum: Peter James Ben Williamson Caroline Ware Nancy Garwood Imperial College: Nigel Bell Jeff Bates Annalisa Massara

1. Introduction

Lichen transplants are widely used to assess the impact of pollution in areas where native thalli are rare or absent. Numerous chemical analytical studies have been carried out including monitoring lead from roads, heavy metals from incinerators and large-scale studies investigating spatial and temporal patterns in metal deposition, as well as assessing the impact of pollutants on various physiological parameters (Garty 2001). The national German guideline VDI 3799 relies on a visual assessment of damage to lichen thalli to evaluate phytotoxic effects. As lichens are notoriously sensitive to environmental change, including local climatic factors, recent guidelines emphasise a need to transplant lichens from sites having ecologically similar environments to those of the transplant sites (Mikhailova 2001).

Monitoring studies in London have identified a decrease in SO2 tolerant species (e.g. Bates et al. 2001). At Burnham Beeches, Buckinghamshire, 40 km to the west of

London, the appearance of species such as SO2 sensitive species such as Xanthoria and the shrubby lichens (Evernia prunastri and Ramalina) were noted on oak trees (Purvis et al. 2001a,b). Photographic monitoring recorded an impact on lichen growth (Parmelia sulcata) during episodic high pollutant emissions, coupled with unusual climatic conditions which suggests that traffic emissions may be responsible for impacts on lichen floras (Purvis et al. 2001a,b). This was supported by chemical analysis of samples demonstrating the accumulation of Zn, N, and other signature elements of traffic pollution. These studies suggested a combination of nitrogen and/or particles may play a role in influencing lichen growth. P. sulcata is one of the most widely employed lichen species in Europe to monitor spatial and temporal patterns in metal deposition.

The Natural History Museum’s Wildlife Estimated concentrations of NOx, NO2 and NO at the Garden situated next 5 sites in the Wildlife Garden, 07/11/01 - 15/03/02 to the junction of 60 Cromwell Road, is 50 infamous for its high 40 NO 30 NO2 traffic density and 20 NOx pollution 10 concentrations (Fig. (ppb) Concentration 0 Site 1Site 2Site 3Site 4Site 5 1). The DEFRA air Monitoring site pollution monitoring station, part of the automatic pollution Fig. 1 recording network is housed in its western corner at the Queen’s Gate intersection (Fig. 2). Kensington and

Chelsea Environmental Health department has also established weekly dust monitoring guages. Regular exceedances of objectives for human health for NO2 (21 ppb annual mean) and particles led the Royal Borough of Kensington and Chelsea to declare the borough as an Air Quality Management Area. Imperial College in association with Newcastle, Bradford and Manchester Metropolitan Universities and the Centre for Ecology and Hydrology (CEH) at Bangor is currently conducting a survey to monitor the effect of urban air pollution on vegetation creating an ideal opportunity for the current pilot project.

2. Objectives

To establish lichen transplants in the NHM wildlife garden in the vicinity of existing N0x,

03, VOC monitoring gauges in order to evaluate the impact of urban air pollution on epiphytic twig communities over a 3-month period:

• To assess the abundance and vitality of 3 lichen groups: fruticose and SO2

sensitive (Ramalina/Evernia) and the foliose Parmelia sulcata (SO2 tolerant) and Xanthoria parietina (N tolerant).

• To sample lichen transplants (Parmelia sulcata and Xanthoria parietina) along the pollution gradient and retain material for future chemical analysis

• To compare results with physicochemical data and make recommendations for future work.

3. Materials and Methods

Lichens on twigs were selected as transplant materials since:-

• Twigs have been successfully used as transplants both to detect temporal and spatial patterns in metal deposition (e.g. Garty 2001).

• Lichen communities on twigs are particularly sensitive to air pollution (Wolseley 1999)

• The majority of thalli on twigs are young, in active growth and therefore less susceptible to senescence through natural causes

• Twigs established on a pole creates a near natural structure where stem flow effects are minimised

Sample collection was carried out during the first week of December 2001 at Nettlecombe, Somerset. Samples were collected from the outer and lower reachable branches of a single isolated Sycamore tree (Acer pseudoplatanus). This species has a neutral/alkaline bark pH and has the closest affinity with the London Plane (Platanus acerifolio) surrounding the Wildlife Garden. Approximately 70 twigs aged between 2-8 years and ca. 40-80cm in length were collected and 50 of these mounted on bamboo radial arms supported on five 7 cm diameter pvc pipes. Other more mature twigs (ca.15) aged between 4-18 years from a different sycamore tree were placed on plastic nets at 3 sites at about 5 m from the road along the southern and western boundary of the wildlife garden (Fig. 2). The winter period was selected for our experiment owing to shading / barrier effect by trees and increased the microclimatic variation during the Summer period. 20 sticky pads were sited adjacent to the trees at each site according to a method developed by Dr Ben Williamson at the Natural History Museum: The garden is situated below the Cromwell and Queens Gate roads:

The Natural

History Museum Fig. 2. Sketch showing Building location of lichen monitoring stations (S1 – S5) and nets (N1 – N3) in the NHM Wildlife Garden

N N3

A N2 N1

Cromwell Road

Monitoring Station Distance from Cromwell road Aspect Site 1 20 m S-facing, exposed Site 2 30m SW, exposed also to Queen’s Gate road Site 3 60m Sheltered by other vegetation Site 4 90m exposed to Queen’s Gate road to the west but sheltered on other sides Site 5 120m S- and W- facing on the north side lies NHM building and Springhouse

Table 1. Transplant site locality details

Changes in the composition of species groups was assessed at the start and end of the transplant period according to the following indices:

Frequency 1 = a single 2 = 1-5 thalli 3 = 6-10 thalli 4=11-20 thalli 5 > 20 thalli Index individual 1 = healthy 2 = slight 3 = moderate 4 = necrotic Vitality index (no signs of signs of signs of (100%) bleaching or necrosis necrosis necrotic (<30%) (>30-50%) lesions) Class size < 1 cm = 1-3 cm = > 3 cm large small ‘s’ medium ‘m’ ‘l’

Table 2. Indices to quantify lichen transplant frequency, vitality and class size

Chlorophyll fluorescence provides a rapid method to determine lichen vitality in the field (Jensen 2002, Jensen & Kricke 2002). Chlorophyll fluorescence was carried out on 12 & 14 January 2001 at the NHM wildlife Garden by Randolph Kricke using a Walz PAM- 210 (teaching PAM) (see http://www.walz.com/pamzhsp.htp. Measurements were repeated using a Hansatech Plant Efficiency Analyser after a 3 month exposure period in the Wildlife Garden (15 March) by Sarah Honour and Annalisa Massara and at the transplant site at Somerset (17 March) by Feliciano Cirimele. Chlorophyll fluorescence was carried out on a minimum of 6 Parmelia sulcata thalli at each station with reference to the parameter Fv/Fm on moistened and previously dark adapted thalli. Dark adaptation was carried out in January using black plastic and in March with dark adaptation clips. Measurements were taken on young margins in all cases.

Sample collection for chemical analysis Samples of P. sulcata and Xanthoria parietina were collected for analysis. Owing to the major loss of P. sulcata towards the centre of the wildlife garden, samples of Evernia and Ramalina were also taken with a view to performing an inter-species calibration.

Fig. 3. Changes in Lichen Frequency as a function of Digital photography thallus size over the period Jan – March 2002

Photographs were taken at Site 1. monthly intervals of 6 thalli of P.sulcata X. parietina E. prunastri+R. farinacea

30 Xanthoria parietina and Parmelia y 25 sulcata at each site using a 20 NIKON Coolpix 995 camera. 15 classes 10

Images were taken at a fixed 5 distance 7 cm using manual Twig's sum of frequenc 0 s-Jan. s-Mar. m-Jan. m-Mar. l-Jan. l-Mar. focus. Size classes: s=small,m=medium, l=large A) 4. Results Site 2. P.sulcata X. parietina E. prunastri=R. farinacea

Lichens at the control site in 20 15 Nettlecombe remained healthy 10 throughout the exposure period, classes 5 0 Twig's sum of Freq. both based on photographic s-Jan. s-Mar. m-Jan. m-Mar. l-Jan. l-Mar. Size classes: small, medium, large recording and measurements of B) chlorophyll fluorescence. Growth Site 3. was recorded in both Parmelia P. sulcata X. parietina E. prunastri=R. farinacea

30 sulcata and Xanthoria parietina 25 20 demonstrating that the lichens 15

classes 10 were successfully transplanted at 5 0 Twigs's sum of Freq. of sum Twigs's Nettlecombe. s-Jan. s-Mar. m-Jan. m-Mar. l-Jan. l-Mar. Size classes: small, medium, large C) Changes in lichen frequency Site 4.

In NHM wildlife garden, an P. sulcata X. parietina E. prunastri+R. farinacea assessment of the frequency of 20 different size classes in the 3 15 species groups revealed different 10 classes trends across the transect (Fig. 5

Twig's sum of Freq. 0 3). Least changes were observed s-Jan. s-Mar. m-Jan. m-Mar. l-Jan. l-Mar in the most exposed sites (1 and Size classes: small, medium, large D) 2) adjacent to road-sites (Figs 3A Site 5.

& B) where fewer losses were P. sulcata X. parietina E. prunastri=R. farinacea recorded than 3 and 4 (Figs 3C & 20

D) (sheltered) and 5 (Fig. 3E) 15 (exposed and furthest from 10 classes 5 roadsides). Maximum losses of Twigs's sum of Freq. 0 all species groups (> occurred in s-Jan. s-Mar. m-Jan. m-Mar. l-Jan. l-Mar. Size classes: small, medium, large large thalli) at site 3 (Fig. 3C), the E) most sheltered site towards the centre of the garden. Virtually all

Parmelia sulcata disappeared from this site. The increase in numbers of small thalli recorded in sites 4 and 5 reflect the disintegration of larger thalli leaving marginal remnants.

Changes in vitality Minor signs of thallus bleaching or the development of a characteristic pinkish or brownish discoloration was observed at any site. Least changes in lichen vitality were recorded at the exposed sites 1 and 2 in all species groups as determined by a visual assessment (Fig. 4) and digital photography. Indeed, images of lichens from Site 2 at the level of Queen’s Gate road showed the least changes.

P. sulcata 3 X. parietina 2 Evernia / Ramalina

0 Vitality index classes 1s 1m 1l 2s 2m 2l 3s 3m 3l 4s 4m 4l 5s 5m 5l Size classes: (small, medium, large) Sites 1, 2, 3, 4, 5

Fig. 4. Average Vitality Index

This contrasted with sheltered sites where major losses occurred. Exposed site 5 furthest from the road suffered the most significant loss of thalli of Parmelia sulcata, particularly towards the inner thallus centre and the site appeared to be deteriorating in a similar fashion to sites 3 and 4. Xanthoria parietina showed the opposite behaviour to Parmelia sulcata with younger thalli suffering the major loss, especially from sites 3 and 4.

Chlorophyll fluorescence measurements for Parmelia sulcata transplants, NHM Wildlife Garden and thalli at Somerset (transplants and native thalli)

0.850

0.800

0.750

Jan-02 Mar-02 Fv/Fm

0.700

0.650

0.600 Native Trans 1 Trans 2 NHM Site 1 NHM Site 2 NHM Site 3 NHM Site 4 NHM Site 5 (Somerset) (Somerset) (Somerset)

Fig. 5

Chlorophyll fluorescence Considerable variation in chlorophyll fluorescence as measured by Fv/Fm was noted (Fig. 5). Most striking is the observation that the Somerset specimens have much lower values (March 2002) than those transplanted to NHM wildlife garden – including measurements made in January 2002 one month after transplantation. The standard error is also considerably larger for March measurements. Measurements confirm that all lichens are physiologically active and alive.

Digital photography Photographic recording confirms that growth has occurred in transplants at the Somerset control site. Little change was noted in thalli at exposed sites at the NHM wildlife garden (1 and 2). Major loss was noted at sites 3 and 4. Older thalli of Parmelia sulcata after 6 weeks of exposure showed a major loss of inner parts at sites 3, 4 and 5. In contrast, larger Xanthoria thalli tend to loose the outer part at the same sites (Appendix E).

5. Discussion

A combination of a visual assessment of abundance, vitality, digital photography and chlorophyll fluorescence confirms that the healthiest lichens are those transplanted at sites 1 and 2 adjacent to the Cromwell and Queen’s Gate roads. Maximum N0, N02, N0x and VOC air concentrations were recorded at these sites suggesting that NOx concentrations which exceed the health standards for human health are not having a

37 major impact on lichen health over the short term (3 months). This contrasts with previous studies where major impacts were recorded on lichens exposed to pollution. Data on the effects of NOx on lichen vitality in the field are lacking although Gilbert (1968) recorded major reductions in chlorophyll content (84%) and respiration (80%) in 3 transplants of the lichen Ramalina farinacea at mean SO2 concentrations of 264 µg/m (Gilbert 1968) during the survey. The loss of lichens in the sheltered sites might be due to a number of reasons quite apart from pollution including:

• Microclimate: Increased shelter will result in prolonged hydration (and hence metabolic activity) during which various pollutants may be in solution. Unfavourable microclimatic factors would result in degeneration in the absence of pollution. December remained dry and relatively warm. Under these conditions lichens would remain physiologically less active. However, rainfall increased during January and February when lichens would be metabolically active and most sensitive to damage from pollution. • Other biological factors including Long-tailed tits and other birds for use in nesting materials, grazing by invertebrates.

Chlorophyll fluorescence Caution is advisable over the interpretation of fluorescence data owing to the different equipment used with potentially different sensitivities, the different operators and methods used to dark adapt thalli. If there is a relationship there appears to be a tendency towards higher levels within the wildlife garden. However, levels above 0.75 are rare in lichens (Jensen 1992) and this upward trend contradicts previous studies mainly examining impacts of SO2 resulting in a decrease in Fv/Fm (see Jensen 2001).

SO2 is well known to result in chlorophyll pigment degradation in lichen photobionts. However, Von Arb & Brunold (1990) identify higher chlorophyll contents in lichens exposed to traffic pollution. There is no significant difference between Fv/Fm values obtained for NHM wildlife garden site 2 (exposed to traffic) and transplant site 1 in Somerset.

Chlorophyll fluorescence vs NO2 concentration

0.850 30

0.800 25 20 0.750 Fv/Fm (March 2002) 15 NO2 (ppb)

Fv/Fm 0.700

10 NO2 (ppb)

0.650 5

0.600 0 Site 1 Site 2 Site 3 Site 4 Site 5

Fig. 6

Chlorophyll fluorescence vs NO concentration

0.850 30

0.800 25 20 Fv/Fm (March 0.750 2002) data 15 NO (ppb)

Fv/Fm 0.700

10 NO (ppb) 0.650 5 0.600 0 Site 1 Site 2 Site 3 Site 4 Site 5

Fig. 7

6. Conclusion

The pilot project confirms that epiphytic lichens on twigs can be successfully introduced into London to study pollution effects even where NO and NO2 levels are high. The absence of a major direct impact on the lichens at road-sites adjacent to Cromwell Rd and Queen’s Gate suggests that NOx is not having a major impact on lichen growth over this time period. This suggests that N may not, at least at these concentrations and meteorological conditions, have an effect on the vitality of P. sulcata although, a longer exposure period would certainly be required to fully test this hypothesis. The possible effect of VOC’s and particulates also needs to be considered. It is interesting to note that the shrubby lichens Evernia and Ramalina remained largely unaffected in this region. There was clearly a varied response amongst the different species and thalli of different ages. We suggest that in addition to pollution, microclimatic factors may have played a role in determining the vitality and loss of particular species. A larger study over a longer time period is required.

7. Recommendations

• It is now important to carry out chemical analysis of the lichen transplants and bark samples we collected during the experiment for N and a range of emission relevant elements. It will be interesting to analyse the lead levels following reductions in the use of leaded petrol. Characterisation of the chemical and physical form of particulates trapped on the sticky pads by SEM and comparison with metal accumulation by lichens at the same sites.

• Further transplant experiments should be established across a wider NOx gradient in London to complement biodiversity studies in areas where insufficient native thalli are available for analysis to determine possible critical levels under different meteorological conditions.

• Further research needs to be carried out to test and develop chlorophyll fluorescence as an indicator of NOx air pollution both in relation to transplants and natural thalli.

• Detailed growth measurements (e.g. Hill 2002) can be carried out on lichens provided sufficient thalli are available for measurements and growth coefficients calculated. In addition to transplants, this can usefully be extended to the study of natural populations as little is understood of the dynamics of natural populations in London.

Appendix A

Estimated Air Pollution Data for the Wildlife Garden, 7th November 2001 to 15th March 2002

Nitrogen Oxides

Concentrations of nitric oxide (NO) and nitrogen dioxide (NO2) at the 5 sites in the Wildlife Garden were estimated by calculating the relationship between measured site concentrations and the NOx levels recorded by the DEFRA monitoring station at the corner of the garden. This relationship was then applied to data from the monitoring site for the time period when the lichen were exposed.

The relationship between NOx concentrations at the sites in the Wildlife Garden and those measured by the DETR monitoring site were calculated from existing data (See

Appendix 1). The NO2 relationship was calculated from 4 sets of measurements made with Palmes type diffusion tubes and 3 sets of measurements made with Ogawa NO/NOx passive samplers. The NO relationship was calculated from 2 sets of measurements made with Ogawa NO/NOx passive samplers. The relationship from the 3rd set of measurements made with these samplers were not used in the calculations since they were taken during a period of high NO concentrations and gave a different relationship to the other measurements. The NO levels recorded at the DEFRA monitoring site during the experiment were similar to those used to estimate the relationship.

DEFRA station Site 1 Site 2 Site 3 Site 4 Site 5 NO % 100 34 32 18 20 20 NO conc.(ppb) 75 25 24 13 15 15

NO2 % 100 67 66 57 52 57

NO2 conc.(ppb) 36 24 24 21 19 21

NOx conc.(ppb) 111 50 48 34 34 36

Figure 1: Estimated NO and NO2 concentrations at the 5 sites in the Wildlife garden, 07/11/01 – 15/03/02

Appendix B

Measurements used to estimate the NO and NO2 relationship for the 5 Sites in the Wildlife Garden

Sampling Site 1 Site 2 Site 3 Site 4 Site 5 Dates DEFR Conc. % Conc. % Conc. % Conc. % Conc. % A (ppb) DEFRA (ppb) DETRA (ppb) DETRA (ppb) DETRA (ppb) DETRA Nitrogen Dioxide Palmes 12-26/07/00 44 27 61 26 59 23 52 20 45 36 type 26/07-23/08 42 33 79 32 76 32 76 27 64 20 48 diffusion 23/08-22/09 44 32 73 34 77 22 50 18 41 tubes 22/09-20/10 47 32 68 26 55 26 55 24 51 29 62

Ogawa 18/10-01/11 37 24 65 24 65 18 49 19 51 18 49 passive 01/11-16/11 39 27 69 27 69 26 67 24 62 27 69 samplers 19/11-3/12 41 23 56 24 59 20 49 20 49 23 56

Relationship 67 66 57 52 57

Nitric Oxide Ogawa 18/10-01/11 82 28 34 27 33 14 17 13 16 16 20 passive 01/11-16/11 103 45 44 39 38 41 40 30 29 33 32 samplers 19/11-3/12 83 28 34 26 31 16 19 20 24 16 19

Relationship 34 32 18 20 20

42 Appendix C

42 Appendix D

43 Appendix E

44 Appendix F

45 Appendix G

46 8. References

Bates, J.W., Bell, J.N.B., & Massara, A.C. (2001) Loss of Lecanora conizaeoides and other fluctuations of epiphytes on oak in S.E. England over 21 years with declining

SO2 concentrations. Atmospheric Environment 35: 2557-2568. Garty, J. (2001) Biomonitoring atmospheric heavy metals with lichens: theory and application. - Critical Reviews in Plant Sciences 20(4): 309-371. Gilbert, O.L. (1968) Biological indicators of air pollution. PhD thesis. University of Newcastle UponTyne. Hill, D.J. (2002) Measurement of lichen growth. In Protocols in Lichenology: Culturing, Biochemistry, Ecophysiology and Use in Biomonitoring. Kranner, I., Beckett, R.P & Varma, A.K., eds.: 255-278, Springer, Berlin. Jensen, M. (2002) Measurement of chlorophyll fluoresence in lichens. In Protocols in Lichenology: Culturing, Biochemistry, Ecophysiology and Use in Biomonitoring. Kranner, I., Beckett, R.P & Varma, A.K., eds.: 136-151, Springer, Berlin. Jensen, M. & Kricke, R. (2002) Chlorophyll fluorescence measurements in the field assessment of the vitality of large numbers of lichen thalli, In Nimis, P.L., Scheidegger, C. & Wolseley, P.A., eds, Monitoring with lichens-Monitoring Lichens Kluwer. Academic Publishers 327-332. Mikhailova, I. (2001) Transplanted lichens for bioaccumulation studies. In Nimis, P.L., Scheidegger, C. & Wolseley, P.A., eds, Monitoring with lichens – Monitoring Lichens. Kluwer, Dordrecht: 301-304. Purvis, O.W., Bamber, R.N., Chimonides, J., Erotokritou, L., Jones, G., Jeffries, T. & Din, V. (2001a) Burnham Beeches Lichen Monitoring Phase 2. Year 3. Report for Corporation of London. Purvis, O.W., Chimonides, J., Din, V.K., Erotokritou, L., Jeffries, T., Jones, G.C., Louwoff, S., Read, H. & Spiro, B. (2002) Which factors are responsible for the changing lichen floras of London? Detecting Environmental Change. Science and Society, 17-20 July, London, UK. Pp 24-25. Purvis, O.W., Chimonides, J, Din, V., Erotokritou, L., Jeffries, T. Jones, G.C., Louwhoff, S., Read, H., Spiro, B. Which factors are responsible for the changing lichen floras of London? Science and the Total Environment [in press] Purvis, O.W., Erotokritou, L., Wolseley, P.A., Williamson, B. & Read, H. (2001) A photographic quadrat recording method employing digital analysis of lichens as an indicator of environmental change. In Nimis, P.L., Scheidegger, C. & Wolseley, P.A., eds, Monitoring with lichens – Monitoring Lichens. Kluwer, Dordrecht: 337-341. VonArb, C. & Brunold, C. (1990) Lichen physiology and air pollution. 1. Physiological response of in situ Parmelia sulcata among air pollution zones within Biel, Switzerland. Canadian Journal of Botany. 68: 35-42.

47 Wolseley, P.A. (2002). Using lichens on twigs to assess changes in ambient atmospheric conditions. - In: Nimis, P.L. et al. (eds.): Monitoring with Lichens – Monitoring Lichens. NATO Science Series, IV, vol. 7. Kluwer, Dordrecht, pp. 291- 294. Wolseley, P.A. & Pryor, K.V. (1999). The potential of epiphytic twig communities on Quercus petraea in a welsh woodland site (Tycanol) for evaluating environmental changes. - Lichenologist 31: 41-61.

SECTION 2

Impacts of urban NOx pollution on vegetation in London

Authors

Sally Power and Tilly Collins Imperial College, London

49 Table of contents

Summary 51 1. Introduction 51 2. Materials and Methods 53 2.1. Transect design and site selection 53 2.2. Calluna sampling 53 2.3. Foliar analyses 53 2.4. Calculation of distance values for transect sites 54 2.5. Estimation of NOx concentrations 54 2.6. The importance of prevailing wind direction 55 2.7. Statistical analyses 56 3. Results and discussion 56 3.1. Relationship between NO2 concentrations and distance 56 3.2. Foliar nitrogen concentration 57 3.3. Carbon:Nitrogen ratios in foliage 58 3.4. Nitrogen : phosphorus ratios 59 3.5. Stable isotope signatures 60 3.6. Trace metals: Lead and Zinc 61 3.7. Implications for urban vegetation. 62 3.8. Estimating total nitrogen deposition inputs 63 4. Conclusions and recommendations 64 5. Acknowledgements 64 6. References 66 Appendix 1 67 Appendix 2 68

Summary

A transect study was carried out during winter 2001/2, to assess changes in shoot chemistry of Calluna (heather) along a gradient of decreasing NOx pollution from central London to rural Surrey. Shoots were analysed for concentrations of nitrogen, carbon, phosphorus, lead, zinc and stable isotopes of nitrogen.

Foliar nitrogen concentrations were positively related to NO2 concentrations and also decreased significantly with increasing distance from central London. Carbon:nitrogen ratios showed the opposite relationship with both NO2 and distance, with linear regressions again statistically significant. High phosphorus concentrations in central London were responsible for a significant increase in foliar N:P ratios with distance from the city centre and suggested that phosphorus availability would not limit plant response to nitrogen at any of the study sites.

NO2 data from the nearest National Diffusion Tube Network sites may not provide the most accurate representation of vegetation exposure to NOx in urban green spaces. Unlike studies elsewhere using mosses, foliar lead and zinc concentrations do not appear to be a useful index of exposure to traffic-derived pollution in Calluna. This may be because the sites used in this study were typically located at distances of more than 150 m from major roads, or because higher plants are more effective at excluding heavy metals than mosses or lichens.

15 Delta N measurements were positively related to NO2 concentrations and declined significantly with increasing distance from central London and increasing distance from major roads. This may suggest a decreasing contribution of traffic-derived NOx to the nitrogen nutrition of higher plants away from busy roads and in less urban areas. However, changes in isotopic signature associated with soil nitrogen transformations and root uptake of nitrate/ammonium make it difficult to interpret this relationship without further investigation of isotopic signatures of nitrogen sources.

Foliar nitrogen concentrations and nutrient ratios suggest that many sites in urban areas experience high deposition inputs of nitrogen and as such are at risk from changes in species interactions and ecosystem function. Further work is required to establish the sensitivity of urban vegetation to realistic levels of both NO and NO2, and to determine the implications of current and future emissions of traffic-derived pollution on urban ecology.

1. Introduction

National air quality standards for gaseous pollutants are concerned primarily with the protection of human health and there are currently only limited data detailing the effects of ambient levels of urban air pollution on sensitive vegetation. Whilst national objectives for NOx and SO2 have been proposed to afford protection to vegetation and ecosystems (DETR, 2001), these are currently excluded from local air quality management plans. There is therefore an urgent need to establish the effects of realistic levels of the principal urban pollutants, NO and NO2, on vegetation in urban areas and, if possible, the contribution that oxidised nitrogen makes to the total deposition load for urban ecosystems. London has the highest levels of traffic-derived pollution in the UK and thus provides a useful starting point from which to assess the potential impacts of NOx on urban ecosystems. Furthermore, the comprehensive network of air quality measurement sites located throughout the capital and home counties provide pollution data in areas of varying traffic density throughout the south-east, and thus a framework within which to assess the effects of NOx on urban ecology.

51 At low concentrations, plants are able to detoxify and assimilate gaseous nitrogen compounds. However, at higher concentrations, foliar damage occurs, often accompanied by changes in physiology, shoot chemistry and detrimental effects on growth. The toxic effects of NO2 are largely associated with its breakdown products, soluble nitrate and nitrite ions (Wellburn, 1990). If concentrations of these ions exceed the cell’s capacity to reduce and assimilate these products, cell damage and/or a disruption in physiology will occur. Effects on photosynthesis, stomatal conductance, foliar chemistry and growth have all been reported following exposure to high concentrations of this pollutant (e.g. Ashenden, 1979; Caporn & Mansfield, 1976; Saxe, 1994).

Whilst NO2 is believed to be considerably more phytotoxic than NO, it has been suggested that NO may also be converted to nitrate and nitrite ions (Wellburn, 1990) and that it may enter the cytoplasm directly (Mansfield, 2001). There is certainly experimental evidence that NO is toxic to plants and that it may have a different phytotoxic mechanism to NO2 (Morgan et al., 1992; Saxe, 1994). Recently it has been shown that NO plays an important role in plant signalling, in particular in relation to plant defence against pathogens (Beligni & Lamattina, 2001; Durner & Klessig, 1999). It is possible, therefore, that high levels of this pollutant may interfere with normal signalling mechanisms.

In addition to the direct toxic effects of NO and NO2, these pollutants also contribute to the total deposition load of nitrogen in urban ecosystems. Since most non-agricultural ecosystems are nitrogen-limited, this may result in a stimulation in growth. Furthermore, increased foliar nitrogen concentrations, changes in phenology and patterns of assimilate distribution have been shown in response to elevated nitrogen availability (See reviews by Bobbink et al., 1998; Green et al., 1997). These changes are associated with increased sensitivity to abiotic and biotic stresses such as drought, frost and herbivore/pathogen attack. Genotypic and phenotypic differences in plant response to both NOx and total nitrogen availability will have an effect on intra- and inter-specific competition and thus, ultimately, on community composition.

Foliar nitrogen concentrations of Calluna vulgaris (heather) have been shown to be related to deposition rates of nitrogen, along gradients of both latitude and altitude (Hicks et al., 1999; Pitcairn et al., 1998, 2001). As such, it Calluna represents a useful indicator species to quantify the magnitude of traffic-derived nitrogen pollution in urban ecosystems. Furthermore, since effects of nitrogenous pollution on the growth and physiology of this species are reasonably well understood, changes in foliar chemistry along a gradient of decreasing pollution will allow a wider evaluation of the potential impacts of NOx on vegetation in urban areas throughout the UK.

It has recently been suggested that oxidised and reduced forms of nitrogen pollution have different natural isotopic signatures; oxidised forms being enriched in 15N, relative to reduced forms (Heaton et al., 1997). The use of δ15N signatures to distinguish between nitrogen sources has been have been pioneered by Pearson et al. (2001), using epilithic mosses which receive most of their nitrogen from the atmosphere. Experiments in The Netherlands have indicated that direct foliar uptake by Calluna shoots can account for up to 60-65% of deposited nitrogen (Heil & Bobbink, 1993). Whilst soil nitrogen transformations and associated changes in isotopic signature may mask any relationship between deposition and foliar δ15N signature, foliar nitrogen concentrations and δ15N signatures of Calluna may nevertheless provide an indication of the contribution of atmospheric pollution to the nitrogen load in urban ecosystems.

The objectives of this pilot study were as follows:

1. To determine whether a relationship exists between foliar nitrogen concentrations of Calluna and ambient NOx concentrations, using a gradient of decreasing pollution away from central London.

2. To evaluate whether isotopic 15N signatures can provide a useful index of the contribution of traffic-derived pollution to the nitrogen nutrition of Calluna.

3. To establish whether plant foliar indicators can provide an estimate of the magnitude of the urban nitrogen footprint.

2. Materials and Methods

2.1. Transect design and site selection A 50 km south-westerly transect was established along a gradient of decreasing atmospheric pollution, from central London to Surrey. The transect thus included 14 sites in either urban, suburban or semi-rural locations; sites ranged from the centrally located Natural History Museum (NHM) Wildlife Gardens to Thursley Common National Nature Reserve, 50 km away in Surrey. Three additional sites in north London, were also sampled. These additional sites were not intended for inclusion in the primary transect, but rather to consider the importance of wind direction (i.e. downwind versus upwind of the metropolitan area) in determining the relationship between location, NO2 data and vegetation response. All statistical analyses presented in the results section are based on the 14 main transect sites and exclude the three north London sites.

Site selection was based on three main criteria: 1) Location along an approximate southwest line from central London; 2) Permission to sample granted by site owners and (where appropriate) English Nature; 3) Sufficient Calluna on site to sample. A map of the transect and details of all sites can be found in Appendix I.

2.2. Calluna sampling At each site (except Barnes Common) three samples of Calluna were taken from distinct, but representative, heathland patches. At Barnes Common, the small patch size of Calluna meant that it was possible to obtain only 2 replicate samples. Sample collection was restricted to current year shoots and was completed during a two week period in winter 2001/2. At the time of collection, shoot growth had ceased for the season. Based on data from The Netherlands (Heil & Bobbink, 1993) and the UK (Power, unpublished data), foliar nutrient concentrations can be expected to have stabilised, at a seasonal low, at this time. At each site, two replicate soil samples were collected, although it was not possible to carry out any analysis on these samples within the scope of this pilot study.

Following collection, plant samples were immediately placed in a chilled environment (cool box) in the field. On return to the laboratory, samples were divided into two; half of the material was frozen at –80oC, while the other half was dried in an oven at 80oC, for three days. Frozen material has been stored for amino acid analysis at a later date and is beyond the scope of the pilot study. Oven dried material was ground to a fine powder using a ball mill.

2.3. Foliar analyses Nitrogen concentration, C:N and N:P ratios. Foliar nitrogen concentrations were determined on dried shoot material. Samples were analysed using a Gas Chromatography Mass Spectrometer (GC-MS), following combustion and oxidation of plant material. Nitrogen concentrations were determined in the Department of Agricultural Sciences at Imperial College (Wye). Carbon concentrations were also determined at this time, allowing an assessment of changes in C:N ratio along the transect.

53 Phosphorus concentrations were determined commercially on dry plant material, by Natural Resource Management Limited, Bracknell, Berkshire (www.nrm.uk.com). Replicate samples were pooled prior to analysis, resulting in a single value per site. Phosphorus is an essential plant nutrient and limits plant growth in many natural ecosystems. Nitrogen:phosphorus ratios have been used as an indicator of plant nutritional imbalance.

Delta 15N. Isotopic enrichment of the heavy (15N) isotope of nitrogen, relative to its lighter (14N), more abundant isotope is increasingly recognised as an indicator of the form of nitrogen which has been assimilated. Isotopic discrimination by many biological processes means that an enrichment of the heavier isotope, relative to its natural abundance in the atmosphere (0.3663 atom %), is indicative of nitrogen uptake predominantly in the oxidised form. This results in a δ15N signature which is positive, with 15 respect to atmospheric N2. Conversely a negative δ N indicates nitrogen uptake predominantly in the reduced form. Isotopic enrichment was analysed, for three replicate samples per site, using a GC-MS. Analyses were carried out in the Department of Agricultural Sciences at Imperial College (Wye). Isotopic enrichment is expressed in parts per thousand, according to the following equation:

15 15 14 15 14 δ N (‰) = (( N: N sample / N: N standard)-1) x 1000

Pb and Zn concentrations: Although lead has not been added to petrol for many years, it has accumulated close to roadsides during past decades. As such, it may provide an useful index of past traffic flow in an area. Zinc on the other hand is a consistent constituent of motor vehicle components and, as such may provide an integrating index of past and current levels of motor vehicle activity in an area. It was thus envisaged that Pb and Zn concentrations might provide information about historical and recent/current traffic flow in the vicinity of each sample site, and thus provide a check on the representativeness of data from the NO2 diffusion tube network to traffic-derived NOx pollution at each of the sites. Foliar Pb and Zn concentrations were measured using atomic absorption spectrophotometry, following digestion of dry plant material in sulphuric acid.

2.4. Calculation of distance values for transect sites Two distance terms for each sample site have been used in the analysis. The first is the straight-line distance from Hyde Park Corner, representing central London; sites were located between 2km and 50 km from the city centre. The second distance term used in the analysis is the distance of the site from the nearest major road (defined as ‘A’ road). Where several A roads were adjacent to a site, the replicate sample locations within a site were measured individually and a mean value obtained.

2.5. Estimation of NOx concentrations

Nitrogen dioxide concentrations at the individual Calluna sampling sites were estimated using diffusion tube data from the UK Nitrogen Dioxide Network. Only sites defined as "background" i.e. >50m from any busy road and typically in a residential area, were used to estimate concentrations at the sample sites. This was to ensure that pollution data came from stations which best reflected the Calluna sampling locations. Since "background" network sites were not always located close to sample sites, consideration was given to averaging data from the nearest roadside and background network sites for those sample sites located within 150 m of the nearest A road. However, since relatively few sample sites were located within this distance, and statistical analysis revealed that this modification did not increase the explanatory power of this variable, only data from background network sites were included in subsequent analyses.

54 Diffusion tube data were used, rather than data from the DEFRA automatic network, because they provide far more detailed spatial coverage of London and the South East, particularly for non-kerb/roadside sites.

Averages of 2000 and 2001 data from the nearest NO2 network site were obtained for each Calluna sampling site. Where a sample site lay between two network sites an average of the concentrations from the two sites was taken. The exception to this was in fact the site at the NHM where measurements of NO2 at the actual sampling location, undertaken as part of a separate study under the NERC-URGENT programme, were used. This provided an accurate reflection of NO2 exposure of Calluna at this site.

Whilst NO2 data from the National Diffusion Tube Network are comprehensive, few data exist for NO concentrations at the same locations. Data from DEFRA monitoring stations around London indicate that in 2000, the ratio of NO to NO2 ranged from 4-5 at the most polluted roadside site (Marylebone Road) to 0.5-1.5 at an urban background site (North Kensington). It seems probable, therefore, that the data obtained from NO2 diffusion tubes at the urban background sites used in this study underestimate NOx concentrations by something in the order of 100%. Additional (unpublished) data, collected by Sarah Honour using paired NO and NO2 diffusion tubes at the NHM Wildlife Garden are summarised in Table 1. These data support the assumption that NO2 data represent only approximately half of the NOx burden in urban areas. This proportion would be expected to decrease with increasing distance from both major roads and the city centre.

Sample Site 1 Site 2 Site 3 Site 4 Site 5 date NO conc. (ppb) 28 27 14 13 16 18/10/01 NO conc. (ppb) 24 24 18 19 18 – 2 01/11/01 NO as a % of 117% 113% 78% 68% 89% NO2 conc. NO conc. (ppb) 45 39 41 30 33 01/11/01 NO conc. (ppb) 27 27 26 24 27 – 2 16/11/01 NO as a % of 167% 144% 158% 125% 122% NO2 conc. NO conc. (ppb) 28 26 16 20 16 19/11/01 NO conc. (ppb) 23 24 20 20 23 – 2 03/12/01 NO as a % of 122% 108% 80% 100% 70% NO2 conc.

Table 1. Summary of NOx data for sites in the NHM Wildlife Garden, 2001. Site 1 is closest to Cromwell Road, while site 5 is furthest away.

2.6. The importance of prevailing wind direction

Concentrations and deposition rates of NOx at a particular site are likely to be influenced by whether the site is upwind or downwind of a given source of pollution (i.e. a major road). In the south of England the prevailing winds are Atlantic south-westerlies. It was therefore considered informative to include a ‘distance moderator’ in the analysis. Such a moderator is based on the assumption that a site downwind of the road in question is effectively closer to it and that one upwind is effectively further from it. This assumption is substantiated by data from the three (downwind) north London sites, which indicated higher deposition loads of nitrogen in relation to distance from central London, than upwind sites. No directional modifier was used for sites parallel to the prevailing wind direction. 55

2.7. Statistical analyses Data manipulation and graph generation were carried out in Microsoft Excel. Linear and multiple regression analyses were performed in Statistica. For all statistical analyses, the 14 sites in the main transect have been used (except where stated). North London sites have been added to graphs for comparison, but are not included in the analysis of main transect data.

3. Results and discussion

In the following analyses, plant parameters have been considered in relation to both NO2 concentrations and distance from central London. This is because NO2 data represent only a proportion of total NOx deposition, whereas distance from the city centre may provide a alternative indicator of the contribution of traffic-derived NOx to the nitrogen load at a given site.

3.1. Relationship between NO2 concentrations and distance

Background concentrations of nitrogen dioxide decline significantly with increasing distance from central London (Figure 1, Table 2).

30.0 Transect 25.0 N London 20.0

(ppb) 15.0 2 10.0 NO 5.0

0.0 0 204060 Distance (km)

Figure 1. The relationship between concentrations of NO2 (ppb) and distance from central London. The north London (downwind) sites are also shown for comparison. Values range from 25.5 µg m-3 in the NHM Wildlife Gardens to 11.4 µg m-3 at Chobham Common in Surrey. The Natural History Museum in South Kensington is located at the junction of the A4 Cromwell Road and Queens Gate, both busy roads with frequent stationary traffic. Due to the fact that NO2 data for this site were obtained within the Wildlife Garden itself, they are considered to accurately reflect the exposure of the sampled vegetation to exhaust emissions and as such, have been retained in subsequent analyses. Interestingly, a statistically significant relationship between distance from the city centre and NO2 concentration is apparent even if this data point is excluded from the regression analysis (Table 2) The three north London sites all lie well above the regression line for the main transect data, with proportionately higher NO2 concentrations for a given distance than the 14 principle sites. Since these three sites are downwind of the urban area, they serve to emphasise the influence of prevailing wind direction. On average the north London sites experience NO2 concentrations 57% higher than would be predicted from the regression equation for the main transect describing the relationship between concentrations and distance from Hyde Park Corner.

56 Relationship d.f. F P r2

Full transect Y= -0.14x + 16.92 1,12 6.45 0.026 * 0.35 Without NHM Y= -0.07x + 14.63 1,11 19.55 0.0013 ** 0.66

Table 2. Summary of the linear relationship between background nitrogen dioxide concentration and distance from central London. The first regression includes all sites along the sampled transect (n=14), the second excludes the Natural History Museum site (n=13).

3.2. Foliar nitrogen concentration

Foliar nitrogen concentrations of Calluna decreased significantly with increasing distance from central London (Figure 2a) and with decreasing background NO2 concentrations (Figure 2b). Values ranged from 1.50% to 0.91%.

2.0 2.0 Transect Sites 1.6 N London Sites 1.6

1.2 1.2 %N 0.8 % N 0.8 0.4 0.4 0.0 Transect Sites

02040600.0 N London Sites 0 5 10 15 20 25 30 Distance (km) (a) NO2 (ppb) (

Figure 2: The relationship between foliar nitrogen concentration and (a) distance from Central London (Hyde Park Corner, London W1) and (b) estimated background NO2 concentration. Error bars represent within-site standard error.

Table 3 summarises the regression analyses for foliar nutrients against distance and NO2 concentrations. It appears that NO2 concentration explains more of the variance in foliar nitrogen than distance, although the relationship was highly influenced by the high NO2 data point from the NHM. Foliar nitrogen concentrations of Calluna do appear to reflect nitrogen deposition in London and the surrounding areas, supporting similar reports in the literature from transect studies across larger geographical and altitudinal gradients (Hicks et al., 1999; Pitcairn et al.,2001).

Relationship with distance from London (km) d.f. F P r2 %N y=-0.0072x+1.26 1,12 6.5 0.025 * 0.35 C:N y=0.28x+40.65 1,12 6.08 0.03 * 0.33 N:P y=0.0059x+10.58 1,12 6.82 0.023 * 0.36

Relationship with background NO concentration 2

%N y=0.034x+0.64 1,12 10.12 0.008 ** 0.46 C:N y=-1.23x+63.79 1,12 7.02 0.021 * 0.37 N:P - 1,12 0.24 0.63 n.s. 0.019

Table 3. Summary of regression analyses for plant nutrient variables against both distance from London and background NO2 concentration.

57 3.3. Carbon:Nitrogen ratios in foliage

As would be expected in view of the relationship with foliar nitrogen concentration described above, carbon:nitrogen ratios in Calluna shoots increased significantly with increasing distance from London (Figure 3, Table 3). Measured ratios ranged from 32 to 56. C:N ratios were also significantly related to NO2 concentrations (Table 3), with the latter explaining a similar proportion of the sample variance as distance.

65 60 55 50 45

C:N 40 35 30 Transect Sites 25 N London Sites 20 0 204060

Distance (km)

Figure 3. Variation in the carbon to nitrogen ratio in the foliage of Calluna with distance from central London (Hyde Park Corner, London W1). Error bars (SEmean) graphically represent within-site variation.

3.4. Nitrogen : phosphorus ratios

Foliar N:P ratios increased significantly with increasing distance from London (Figure 4, Table 3), contrary to expectations. The relationship between N:P ratios and NO2 concentration was, however, not significant. It appears that phosphorus availability is higher in urban than in rural areas. Using the N:P ratio as an index of relative nutrient limitation (Koerselman & Mueleman, 1996), the data suggest that none of the sites are currently phosphorus-limited and thus may be expected to continue to exhibit growth and other responses to nitrogen inputs in the future.

15 14 13 12 11 10 N:P 9 8 Transect Sites 7 6 N London Sites 5 0204060 Distance (km)

Figure 4. Variation in the nitrogen to phosphorus ratio in the foliage of Calluna with distance from central London.

58 3.5. Stable isotope signatures

15 The relationship between δ N and background NO2 concentration was statistically significant, indicating a decline in the relative proportion of the heavy (15N) isotope with decreasing NO2 concentrations (Figure 5, Table 4). The relationship was, however, driven by the high (but accurately measured) value for NO2 at the NHM, and explained a relatively small proportion of the data variance.

NO2 0102030 (ppb) 5 N

15 0

-5 Delta Delta -10 Transect Sites N London Sites

15 Figure 5. Relationship between δ N signature and background NO2 concentration. Error bars represent within-site standard error.

Since NO2 concentrations represent a smaller proportion of total oxidised nitrogen in urban areas, particularly close to roads, than in rural areas, the relationship between isotopic signature and distance from both central London and the nearest major road was also investigated. Table 4 summarises the linear regressions for both parameters.

d.f. F P r2

NO2 concentration y=0.43x-9.91 1,12 5.92 0.031 * 0..33

Distance from London y=-0.12x-1.44 1,12 9.62 0.009 ** 0.44

15 Table 4. Summary of linear regressions for relationship between δ N and either NO2 concentration or distance from London

A highly significant negative relationship was seen between δ15N and distance from central London (Figure 6a). A similar, relationship, albeit with more scatter, was also seen for increasing distance from major roads (Figure 6b).

Transect Sites Transect Sites Distance 1 (km) Distance 2 (km) N London Sites N London Sites

0 102030405060 0 0.2 0.4 0.6 0.8 1 1.2 2 2 0 0 N -2 N -2 15 15 -4 -4 -6 Delta -6 Delta -8 -8 (a) -10 (b) -10

Figure 6. The relationship between δ15N signature and (a) Distance 1(km from Hyde Park Corner) and (b) Distance 2 (km from the nearest A road, moderated by -50% if downwind and +50% if upwind)

59 The contribution of both distance parameters to the variation in foliar δ15N values for Calluna was also investigated using multiple regression analysis. This revealed that both distance variables contributed significantly to an explanation of the variance in foliar δ15N (Table 4).

Parameter SE t p

estimate Intercept -0.51 0.94 -0.55 0.59 n.s. Distance 1 -0.093 0.036 -2.60 0.025 * Distance 2 -3.59 1.62 -2.21 0.049 *

2 Full model: r =0.62 F2,11=8.82 P=0.0052 **

Table 4. Multiple regression summary table of δ15N signature with Distance 1 (distance in km from Hyde Park Corner) and Distance 2 (distance in km from the nearest A road, moderated by -50% if downwind and +50% if upwind). The relationship with distance from central London was in the direction expected from earlier work by Pearson et al. (2000). The latter study reported a positive δ15N signature in epilithic mosses at kerbside sites, falling to a negative signature with an increasing contribution of reduced nitrogen to total deposition. δ15N values for Calluna growing close to the roadside at the Natural History Museum were positive, although, none of the other sites were within 100 m of a major road.

However, is difficult to interpret these data without investigation of the isotopic signature of pollutant sources in central London. Ammonia emissions associated with catalytic converters can be expected to be relatively high at kerbside sites. Since NH3 has a higher deposition velocity than NO2, reduced nitrogen may in fact make a substantial contribution to nitrogen inputs in kerbside areas. There is clearly a great need to investigate the isotopic signatures of vehicle-derived NOx and NH3, and the effect of soil nitrogen transformations on δ15N signatures, before any conclusions can be reached concerning the use of this parameter as an index of traffic-derived pollution. Nevertheless, a significant negative relationship between δ15N and distance from central London was found in this investgation and suggests that this relationship may be worth pursuing in a more detailed follow on study.

The δ15N data from the current study is unable to reveal information about the proportion of foliar (as opposed to root) uptake of nitrogenous pollution for Calluna in urban areas. A variety of soil, plant and environmental factors affect the δ15N signal of foliage in higher plants. Högberg (1997) lists these factors as (1) the source of nitrogen (soil, precipitation, NOx, NH3, N2 fixation), (2) the depth(s) in soil from which nitrogen is taken up, (3) the + - forms of soil-N used (organic N, NH4 , NO3 ), (4) influences of mycorrhizal symbioses and fractionations during and after nitrogen uptake by plants, and (5) interactions between these factors and plant phenology.

3.6. Trace metals: Lead and Zinc The study by Pearson et al. (2000) mentioned above also suggested that lead and zinc concentrations in moss tissue may provide a reasonable index of traffic density and thus, by extrapolation, of exhaust emissions in an area. Although lead is no longer added to petrol, high concentrations in the soil adjacent to formerly busy roads, together with a re- circulation of Pb-contaminated dust and soil particles near roadsides might be expected to lead to a legacy of enhanced foliar concentrations. Elevated concentrations could therefore reflect past and possibly present (in proportional terms) traffic flow in a given area. A similar pattern can also be expected for zinc, since in urban areas, away from smelters and heavy industry, the main sources of zinc are likely to be wear of vehicle parts, particularly engines and tyres (Freedman & Hutchinson, 1981). 60

The highest foliar concentrations of lead were, somewhat surprisingly, found at Chobham Common in Surrey, whereas zinc was highest on Hampstead Heath. Whilst the latter site is relatively close to a major road, the locations from which shoots were collected at Chobham Common were not. No significant relationship was found between either of the heavy metals and distance from nearest A road (Figure 7), distance from central London, or background NO2 concentrations.

16 16 14 14 12 12 10 10 Pb ppm 8 Zn ppm 8 6 6 4 4 <=200m >200m,< >600m <=200m >200m,< >600m a 600m b 600m

Figure 7. Shoot lead (a) and zinc (b) concentrations in Calluna averaged over three distance categories from the nearest A road. Error bars show the SE. Overall, measured concentrations were very low (5-15 ppm Pb, 12-15 ppm Zn); more than an order of magnitude lower than those reported for moss in Pearson et al. (2000). Several reasons may exist for this. Firstly, Pearson et al. (2000) reported that heavy metal concentrations fall off rapidly at distances of greater than 40m from roadsides. With the exception of the Natural History Museum, all of the sites used in this study were further than this from major roads. Secondly, a greater degree of discrimination against uptake of heavy metals would be expected for mycorrhizal plants such as Calluna, compared to mosses. Mycorrhizal fungi have been suggested to reduce heavy metal uptake by acting both as physical barriers to uptake and by precipitation/complexation in the hyphal network (e.g. Wilkins, 1991). It would appear that neither Pb nor Zn are useful surrogates for NOx exposure in higher plants.

3.7. Implications for urban vegetation.

Nitrogen dioxide diffusion tube data suggest that, amongst the sites used in this study, critical levels for NOx are exceeded only at the NHM Wildlife Gardens in South Kensington. This, however, contrasts with maps showing modelled NO2 data across greater London, which suggest that large areas of the capital exceed the 16 ppb threshold for vegetation effects (GLA map reproduced in Figure 1.1 in the previous section of this report). Consideration must also be given to the fact that a lack of appropriate data for NO led to its exclusion from this study. Whilst the contribution of NO to NOx declines with increasing distance from roadsides, measurements in the NHM Wildlife Gardens (Honour, unpublished data) indicate that NO concentrations approximately equal those for NO2. Data from the DEFRA automatic monitoring station at Harwell suggest NO:NO2 ratios in the region of 0.23 (2001) to 0.47 (2000). NOx levels are therefore undoubtedly higher in urban areas than indicated by NO2 diffusion tube data alone, suggesting that NOx standards might be exceeded at many more of the study sites.

Plant responses to nitrogen deposition are varied, but include a number of potentially detrimental effects on physiology, growth and phenology. A reduction in fine root biomass, root:shoot ratios and an increased sensitivity to drought have been shown for many ecosystems (e.g. De Visser et al., 1996; Bardgett et al., 1999; Power et al., 1998; Gorden et al., 1999). This may be particularly important for vegetation in urban environments which typically experience higher air temperatures and lower humidities than rural areas. 61

Changes in plant phenology and associated effects on sensitivity to low temperature stress have also been linked to enhanced nitrogen availability. For example, earlier spring growth of Calluna and an associated increase in sensitivity to frost stress has been demonstrated in both field and laboratory studies (Gorden et al., 1999, Power et al., 1998, Van der Eerden et al., 1991). Increased foliar nitrogen concentrations and decreased C:N ratios are frequently reported in response to elevated nitrogen availability. This study showed a clear relationship between these variables and both distance from central London and NO2 concentration. Furthermore, stable isotope signatures indicate that vehicle-derived pollution constitutes a greater proportion of the nitrogen budget of plants in inner city, compared with rural areas. Elevated foliar nitrogen concentrations are typically associated with increased attack by insects such as aphids (Fluckiger & Braun, 1998) or by fungal pathogens (Strengbom, 2002).

Species differences in growth response to elevated nitrogen inputs and sensitivity to biotic and abiotic stresses will affect resource competition at a community level, and there is a strong indication from this study that urban ecosystems may be more at risk from nitrogen-induced changes in species composition and associated effects on ecosystem function and biodiversity than vegetation in more rural areas. Changes in plant growth and community composition have been reported with increasing proximity to a major trunk road in the UK (Angold, 1997). Angold (1997) suggested that changes in the growth and vitality of lichens and higher plants in the New Forest could be related to vehicle emissions, providing field evidence of the potential for effects of NOx on sensitive plant communities.

3.8. Estimating total nitrogen deposition inputs

As mentioned previously, foliar nitrogen concentrations in Calluna have been shown to have a distinct relationship with nitrogen deposition load across wide geographical ranges and across a variety of soil types (Hicks et al., 1999; Pitcairn et al., 1998, 2001). In their recent study involving data from both the UK and NW Europe, Pitcairn et al. (2001) reported that foliar nitrogen concentrations increased at a rate of 0.036% for every 1 kg ha-1 yr-1 increase in nitrogen deposition rate, with an intercept of 0.865 %N. If this relationship is applied in reverse, total nitrogen deposition loads can be estimated from foliar concentrations.

The lowest foliar concentrations found in this study was 0.91% N, at Thursley Common, 50 km away from central London. The equation derived by Pitcairn et al. (2001) indicates that this represents a deposition load of approximately 1.25 kg N ha-1 yr-1. This is in fact considerably lower than calculated total nitrogen deposition for this site (10-15 kg ha-1 yr-1; Power, unpublished data) and may be in part be explained by the fact that sample collection for this study took place at a time when foliar nitrogen concentrations are at a seasonal low (Heil & Bobbink, 1993) whereas the relationship established by Pitcairn et al. (2001) used concentration data obtained during the summer/autumn, when levels are typically higher. Foliar nitrogen concentrations have been measured at different times of the year at Thursley Common, as part of an ongoing study, and show that summer concentrations are typically 25% higher than those measured in autumn/winter (S. Power, unpublished data). If a seasonal correction is applied to data for this site, winter values of 0.91% N could be considered equivalent to summer values of 1.14 %. Using the Pitcairn et al. (2001) relationship, this would equate to a deposition load of 7.6 kg N ha-1 yr-1.

The highest foliar concentrations measured in the present study were 1.5% N, at the Natural History Museum. Using the same relationship, this represents a deposition load of 17.6 kg N ha-1 yr-1. When corrected for seasonality, this rises to values of 1.88% and 28.0 kg N ha-1 yr-1 respectively. Another way of looking at the relationship between foliar nitrogen concentration and deposition is to establish a threshold concentration which represents exceedance of a particular deposition load. For Calluna-dominated heathland the critical load for nitrogen is 15-20 kg ha-1 yr-1. Using the Pitcairn et al. (2001) 62 relationship, this equates to summer/autumn foliar concentrations of 1.33-1.58%. Given the seasonal differences in foliar concentrations, it seems reasonable to suggest that sites with winter foliar concentrations in excess of 1.06-1.26% N (equivalent to 15-20 kg ha-1 yr-1, after applying a 25% seasonal correction to the winter foliar %N values) receive nitrogen inputs in excess of current critical loads for this nutrient.

The Natural History Museum, Barnes Common and Coombe Hill had (winter) foliar nitrogen concentrations of 1.5%, 1.44% and 1.27% (respectively), suggesting that these sites receive nitrogen deposition loads in excess of 20kg ha-1 yr-1. Critical nitrogen loads have been suggested for only a limited number of ecosystem types (Bobbink & Roelofs, 1995). However, with the exception of particularly sensitive ecosystems which would not occur in UK urban areas (e.g. ombrotrophic fens, arctic/alpine heaths), most suggested critical loads fall within the 15-30 kg N ha-1 yr-1 range. It seems likely, therefore, that in addition to exceeding critical levels for NOx, many urban ecosystems are also receiving deposition inputs close to or in excess of the nitrogen critical load.

One of the aims of this study was to try to assess the magnitude of the urban nitrogen footprint. However, this has not been possible for a number of reasons. Of the possible indicators of urban nitrogen deposition (%N, C:N, δ15N), none showed a clear cut off at a particular distance from the city centre. There was an indication that %N was higher and C:N ratio lower in inner city than in suburban areas, but there are insufficient data points to fit a non-linear slope to the graph with distance for these variables. δ15N also had the potential to be an indicator of the size of the urban nitrogen influence, but was clearly affected by site location in relation to major roads, and possibly also root uptake of nitrogen. An increase in the number of sites surveyed and the inclusion of other species such as mosses (which were initially proposed, but outside of the scope of this pilot study), could provide a clearer estimate of the geographic extent of traffic-derived pollution in urban/suburban areas.

4. Conclusions and recommendations

Whilst this study was intended as only a small scale pilot project, it has nevertheless generated some important data which indicate that urban ecosystems are experiencing considerably higher deposition inputs of nitrogen than those in more rural locations. Foliar nitrogen concentrations are particularly high in central London and, whilst NO2 represents only a proportion of total oxidised deposition in urban areas, these parameters were significantly related. δ15N values for Calluna shoots were related to distance from central London, although it is not currently possible to interpret this as an index of traffic-derived pollution without further study. Investigation of the isotopic signature of traffic- derived NOx and NH3, together with more detailed assessment of changes associated with soil N transformations and root uptake in urban areas would enable an evaluation of the use of isotopic signatures in higher plants as biological indicators of traffic pollution.

Although the pilot project was not designed to test whether plants exposed to higher concentrations of NOx exhibit changes in growth or phenology, the implications are that nitrogen deposition loads are high enough to have effects on species composition and ecosystem stability. However, an assessment of the sensitivity of urban vegetation to ambient levels of NO2 and, perhaps even more importantly, NO is urgently required. Large cities are subject to a variety of environmental stresses, such as higher temperatures and wind speeds. These, in combination with other factors unique to built up areas (e.g. night time illumination from street lights) may make urban vegetation particularly sensitive to atmospheric pollution. Interactions between atmospheric pollution and these urban stresses therefore clearly require investigation.

63 The current study demonstrated the value of Calluna as an indicator species but, of necessity, was restricted to remnants of acidic heathland habitat within the London area. This naturally limited the choice of sites and resulted in the use of samples at a variety of distances from major roads. An expansion of this pilot study, to include further sites and other key species, would allow a more accurate assessment of the contribution of local, as opposed to diffuse, sources of NOx and the applicability of the results obtained for Calluna to other vegetation types and habitats.

5. Acknowledgements

The contribution Sarah Honour made to this report, particularly in terms of pollution data from the National Diffusion Tube Network and from her own studies at the Natural History Museum's Wildlife Gardens, was invaluable. Her help is greatly appreciated and gratefully acknowledged. Cathy Shields, Jon Fear and Melanie Hardie all helped with sample preparation and chemical analyses at speeds beyond the call of duty, making it possible to produce this report in the available timeframe, so my thanks also to them. Thanks are also due to the many site owners, wardens and managers who not only gave permission to sample, but who often enthusiastically accompanied me on sample visits.

6. References

Angold, P.G. 1997. The impact of a road upon adjacent heathland vegetation: Effects on plant species composition. Journal of Applied Ecology, 34, 409-417. Ashenden, T. (1979) Effects of SO2 and NO2 pollution on transpiration in Phaseolus vulgaris L. Environmental Pollution 18, 45-50. Bardgett, R., Mawdsley, J., Edwards, S., Hobbs, P., Rodwell, J. and Davies, W. (1999) Plant species and nitrogen effects on soil biological properties of temperate and upland grasslands. Functional Ecology 13, 650-660. Beligni, M. and Lamattina, L. (2001) Nitric oxide in plants: the history is just beginning. Plant Cell and Environment 24, 267-278. Bobbink, R., Hornung, M. and Roelofs, J. (1998) The effects of air-borne nitrogen pollutants on species diversity in natural and semi-natural European vegetation. Journal of Ecology 86, 717-738. Bobbink, R. and Roelofs, J. (1995) Nitrogen critical loads for natural and semi-natural ecosystems: The empirical approach. Water, Air & Soil Pollution 85, 2413-2418. Caporn, S. and Mansfield, P. (1976) Inhibition of net photosynthesis in tomato in air polluted with NO and NO2. Journal of Experimental Botany 27, 1181-1186. DETR (2000) The air quality strategy for England, Scotland and Wales. 2000. Department of the Environment, Transport and the Regions, London. DeVisser, P., Keltjens, W. and Findenegg, G. (1996) Transpiration and drought resistance of Douglas-fir seedlings exposed to excess ammonium. Trees-Structure and Function 10, 301-307. Durner, J. and Klessig, D. (1999) Nitric oxide as a signal in plants. Current Opinion in Plant Biology 2, 369-374. Flückiger, W. and Braun, S. (1998) Nitrogen deposition in Swiss forests and its possible relevance for leaf nutrient status, parasite attacks and soil acidification. Environmental Pollution 102, 69-76. Freedman, B. & Hutchinson, T.C. (1981) Sources of metal and elemental contamination of terrestrial environments. In: Effect of heavy metal pollution on plants (ed. N.W. Lepp). Applied Science Publishers, London 35-94. Gordon, C., Woodin, S., Alexander, I. and Mullins, C. (1999) Effects of increased temperature, drought and nitrogen supply on two upland perennials of contrasting functional type: Calluna vulgaris and Pteridium aquilinum. New Phytologist 142, 243-258

64 Green, P.R.S., Power, S.A., Ashmore, M.R. and Bobbink, R. (1997) Whole ecosystem nitrogen manipulation: review study. English Nature, Peterborough. 68 pp. Heaton, T. H. E., Spiro, B., Madeline, S. and Robertson, C. (1997) Potential canopy influences on the isotopic composition of nitrogen and sulphur in atmospheric deposition. Oecologia 109, 600-607. Heil, G. W. and Bobbink, R. (1993) Impact of atmospheric nitrogen deposition on dry heathlands. In Heathlands: Patterns and processes in a changing environment, (eds. R. Aerts and G. W. Heil), pp. 181-200, Kluwer Academic Publishers, Dordrecht. Hicks, W.K., Leith, I.D., Woodin, S.J., & Fowler, D. 2000. Can the foliar nitrogen concentration of upland vegetation be used for predicting atmospheric nitrogen deposition? Evidence from field surveys. Environmental Pollution, 107, 367-376. Koerselman, W. & Mueleman, A.E.M. (1996) The vegetation N:P ratio : a new tool to detect the nature of nutrient limitation. Journal of Applied Ecology 33, 1441-1450. Mansfield, T. (2001) Nitrogen oxides: Old problems, new challenges. In Air pollution and plant life, (eds. J. Bell and M. Treshow), In Press. Morgan, S.M., Lee., J.A. et al. 1992. Effects of nitrogen oxides on nitrogen assimilation in bryophytes. New Phytologist, 120, 89-97 Pearson, J., Wells, D. M., Seller, K. J., Bennett, A., Soares, A., Woodall, J. and Ingrouille, M. J. (2000) Traffic exposure increases natural N-15 and heavy metal concentrations in mosses. New Phytologist 147, 317-326. Pitcairn, C. E. R., Fowler, D. and Grace, J. (1995) Deposition of fixed atmospheric nitrogen and foliar nitrogen-content of bryophytes and Calluna vulgaris (L) Hull. Environmental Pollution 88, 193-205. Pitcairn, C. E. R., Leith, I. D., Fowler, D., Hargreaves, K. J., Moghaddam, M., Kennedy, V. H. and Granat, L. (2001) Foliar nitrogen as an indicator of nitrogen deposition and critical loads exceedance on a European scale. Water Air and Soil Pollution 130, 1037-1042. Pitcairn, C. E. R., Leith, I. D., Sheppard, L. J., Sutton, M. A., Fowler, D., Munro, R. C., Tang, S. and Wilson, D. (1998) The relationship between nitrogen deposition, species composition and foliar nitrogen concentrations in woodland flora in the vicinity of livestock farms. Environmental Pollution 102, 41-48. Power, S. A., Ashmore, M. R., Cousins, D. A. and Sheppard, L. J. (1998) Effects of nitrogen addition on the stress sensitivity of Calluna vulgaris. New Phytologist 138, 663-673. Saxe, H. (1994) Relative sensitivity of greenhouse pot plants to long-term exposures of NO-containing and NO2-containing air. Environmental Pollution 85, 283-290. Strengbom., J. (2002) Nitrogen, parasites and plants - key interactions in boreal forest ecosystems. PhD Thesis, University of Umeå, Sweden. Van der Eerden, L., Dueck, T., Berdowski, J., Greven, H. and Van Dobben, H. (1991) Influence of NH3 and (NH4)2SO4 on heathland vegetation. Acta Botanica Neerlandica 40, 281-296. Wellburn, A. R. (1990) Why are atmospheric oxides of nitrogen usually phytotoxic and not alternative fertilizers. New Phytologist 115, 395-429. Wilkins, D. (1991) The influences of sheathing (ecto) mycorrhizas of trees on the uptake and toxicity of metals. Agriculture, Ecosystems and Environment 35, 245-260.

65 16 17 15

2 13 4 8 5 12 6 9 7 10

14 11

Appendix 1. Map of the sample transect (1-14) and North London sites (15-18)

Sample transect sites, their distances from Hyde Park Corner and the nearest ‘A’ road No Site name Distance from Distance from Hyde Park Corner nearest ‘A’ road (km) (km) 1: Natural History Museum 2 0.02 2: Barnes Common 7 0.10 3: Hampstead Heath 7.5 0.03 4: Putney Heath 9.5 0.34 5: Wimbledon Common 10.5 0.63 6: Richmond Park 13 0.90 7: Coombe Hill Golf Course 13.5 0.57 8: Hounslow Heath 17.5 0.53 9: Esher Common 23.5 0.20 10: St Georges Golf Course, Byfleet 27 0.57 11: Chobham Common 35 0.44 12: Horsell Common 36 0.56 13: Silwood Park 37.5 0.17 14: Thursley Common 50.5 1.00

North London Sites 15: Ruislip 22 0.33 16: Stanmore Common 18.5 0.26 17: Epping Forest 24.5 0.20

66 Appendix 2: Summary of estimated annual mean NO2 concentrations for Calluna sites

National Diffusion Tube Distance from Calluna sampling site Monitoring Network site Calluna site 2000 2001 Average (km) (ppb) (ppb) (ppb) Natural History Museum Natural History Museum 0 30 21 25.5 Barnes Common KENSINGTON 4N 4.3 16 12.7 RICHMOND UPON Barnes Common THAMES 3N 2.3 13.3 13.2 Barnes Common Average 14.7 13.0 13.8 Hampstead Heath CAMDEN 6N 1.3 14.5 15.2 14.5 Putney Heath KENSINGTON 4N 6.9 16 12.7 RICHMOND UPON Putney Heath THAMES 3N 4.2 13.3 13.2 Putney Heath Average 14.7 13.0 13.8 Wimbledon Common KENSINGTON 4N 8.1 16 12.7 RICHMOND UPON Wimbledon Common THAMES 3N 4.9 13.3 13.2 Wimbledon Common 14.7 13.0 13.8 RICHMOND UPON Richmond Park THAMES 3N 4.0 13 13.2 13.1 RICHMOND UPON Coombe Hill THAMES 3N 5.3 13 13.2 13.1 SUNBURY ON THAMES Hounslow Heath 1N 4.8 14.1 14.2 RICHMOND UPON Hounslow Heath THAMES 4N 5.0 13.7 13.2 Hounslow Heath Average 13.8 13.8 13.8 Esher Common ESHER 3N 2.3 12.6 14.8 13.7 Byfleet ADDLESTONE 5N 3.5 13.1 13.8 13.5 Chobham WOKING 7N 6.3 11.5 Chobham ADDLESTONE 3N 3.6 13.1 9.6 Chobham Average 12.3 9.6 11.0 Horsell Common ADDLESTONE 3N 3.6 13.1 9.6 11.4 Silwood Park WINDLESHAM 1N 4.5 14.0 Silwood Park WOKING 7N 9.9 11.5 Silwood Park Average 11.5 14.0 12.8 Thursley Common FARNHAM 4N 8.0 11.8 8.7 Thursley Common GUILDFORD 3N 13.4 11.2 14.0 Thursley Common Average 11.5 11.3 11.4

Ruislip RICKMANSWORTH 3N 5.2 18.8 18.8 18.8 Stanmore Common BOREHAMWOOD 4N 4.5 23.3 16.8 20.1 Epping Forest BOREHAMWOOD 4N 5.3 23.3 16.8 20.1

SECTION 3

Assessing the Role of Biological Monitoring Using Lichens to

Map Excessive Ammonia (NH3) Deposition in the UK

Authors Pat Wolseley & Peter James The Natural History Museum London

Acknowledgements The authors would like to thank the following for assistance with this project: Roger Smith and others at IGER, North Wyke for supplying maps and site information, Mark Sutton and Tristan Bantock at CEH Edinburgh and Zandra Waterford of Breckland Council for Environmental Health for supplying ammonia data and maps of sites, MOD for access and transport to military ranges for survey in the Thetford area, Robert Abrey for providing access to trees on farmland and Bev Nicholls for access and maps to the Norfolk Wildlife Trust reserve at Wretham heath.

68 1. Background 70 2. Objectives 70 3. Method 70 3.1 Site Selection 70 3.2 Station selection 71 3.3 Lichen recording 71 3.4 Lab techniques 71 3.5 Data Analysis 71 4. Results 72 5. Discussion 77 6. Conclusions 79 7. Recommendations 79

Appendices A. Maps of lichen sampling stations at Thetford and North Wyke. 80 B. Table of NIW and AIW species according to van Herk (1999,2002) 81 C. Table of epiphytic lichens on Quercus trunks near Thetford, Norfolk 82 D. Table of epiphytic lichens on Quercus trunks at North Wyke, Devon 83 E. Table of species recorded on Quercus twigs. 85

69 1. BACKGROUND

Research on isolated oak trees in the vicinity of intensive livestock units in the Netherlands has shown a strong correlation between the composition of lichen communities and atmospheric ammonia levels (van Herk 1999, 2002). The recording technique identified species that were obligate nitrophytes (nitrogen-loving species) or acidophytes (SO2 tolerant species) enabling the construction of indices of nitrophyte ‘NIW’ (Nitrofiele Indicatie Waarde) and acidophytes ‘AIW’ (Acidofiele Indicatie Waarde).

In Britain, the Ammonia monitoring network at the Centre for Ecology and Hydrology, Edinburgh (http://www.nbu.ac.uk/cara/UKNAMN/uknamn.htm), monitored ammonia levels at 95 sites. Recent research in the vicinity of intensive poultry units in Britain has shown high levels of ammonia deposited locally along a deposition gradient of up to c. 300m from source (Fowler et al., 1998). The residence time of ammonia in the atmosphere is rather brief so that the effects on vegetation are often localised around the source (NEGTAP 2001).

Although lichens have long been shown to be indicators of atmospheric acidification (Nimis et al., 2002), recent observations on epiphytic lichen communities in rural areas in the Netherlands have shown a widespread change in lichen communities with an increase in nitrophytes and a corresponding decrease in acidophytes (van Herk, 1999). Whereas there is little variation in the climate of the Netherlands, there is a marked climatic gradient across Britain ranging from drier conditions in the east to wetter oceanic conditions in the west which have a pronounced influence on the species composition of lichen communities. The continental method developed by van Herk was applied during the present survey in the contrasting climatic regions of Norfolk and Devon, in the vicinity of ammonia monitoring stations in order to test the potential application of the system in Britain.

2. OBJECTIVES

• To collect data from 2 sites in Britain with a history of intensive livestock management

where NH3 emissions have been monitored over a period of time. • To test the results against scales developed in the Netherlands. • To evaluate the method in order to develop a biomonitoring procedure in Britain.

3. METHOD

3.1. Site selection Sites were selected using aerial photographs, taking into account: the availability of ammonia monitoring stations; the distribution of lichens in Britain including both continental and oceanic components; the presence of exposed oak trees on acid soils; and the presence of a site of conservation importance in the vicinity.

A site with a continental climate was selected in the Thetford region, Norfolk, where extensive ammonia monitoring has been taking place in the vicinity of intensive poultry 70 and pig rearing units and in unimproved grassland utilised by the MOD. A site with an oceanic climate at North Wyke in Devon was chosen where ammonia is recorded in the vicinity of intensive cattle rearing sheds at IGER (Institute of Grassland and Environmental Research).

3.2. Station selection Stations were selected according to availability of Quercus trees and prevailing wind direction. The distance to source of pollution was estimated from ordnance survey maps. Managed woodland and woodland edge trees were avoided where possible, as lichen communities varied considerably in these sites.

Stations were selected in Norfolk to coincide with existing ammonia monitoring stations. At North Wyke stations were selected within zones of up to 1000m radius of the farm units, as the ammonia station is located south and upwind of the cattle sheds and slurry tank. Given the restrictions of time and the availability of trees, 3-5 upright standard Quercus trees >50cm girth were selected in exposed well-lit situations. Leaning trees were avoided or those with low epicormic growth or ivy on the trunk. Trees on the edge of managed woodland or intensively used roads were also avoided. Stations and tree position were marked on a map. Bark samples were collected for pH determination.

3.3. Lichen recording. Corticolous species on each tree trunk up to 2m were listed and assigned a frequency value according to van Herk; 1 thallus on the trunk, 2 or more thalli, and species with >10cm2 present. Where identification was not possible in the field specimens were collected for identification in the laboratory. The data was subsequently converted for each station according to van Herk. a) Only one thallus present b) 2 or more thalli on one tree c) Present on 30-50% of trees, < 10cm2/tree d) Present on 30-50% of trees, > 10cm2/tree e) Present on 50-100% of trees, <10cm2/tree f) Present on 50-100% of trees, >10cm2/tree g) NIW and AIW species were distinguished and scored according to van Herk and the mean AIW and NIW scores for each station were calculated (Appendix 1).

3.4. Lab techniques Bark pH. 3 samples of bark were collected from each tree, placed in paper packets and dried. pH was measured using a GELPLAS flathead electrode after moistening bark with 25% KCl for c. 5 minutes (Bates et al. 1990). Lichen species were identified using light microscopy and thin layer chromatography

3.5. Data analysis Ammonia deposition was supplied as monthly averages for all stations in the Thetford area by the Breckland Council for Environmental Health and by CEH Edinburgh ammonia 71 for the period February to October 2001 and as monthly averages for North Wyke by CEH Edinburgh between May 1997 and September 2001. Where present, mean ammonia values for lichen sampling stations in the Thetford area were plotted against NIW and AIW indices. As ammonia deposition was only recorded at one station at North Wyke, the distance from the farm units and slurry tank was plotted against NIW and AIW indices as a surrogate measure for ammonia deposition.

4. RESULTS

Site 1: Thetford area, Norfolk In Norfolk 12 stations were sampled in the Thetford area coinciding where possible with ammonia monitoring stations (Fig1, table 1). Table 1. Stations in the Thetford area.

NH3 deposition Lichen diversity Stat Station location NH3 High Mean Low no. Trunk Twig Distance from Direction from no. T’s source source 1 Norfolk Wildlife Reserve 1 11 2.64 2.88 0.8 5 7.4 5 500 SW 2 Abrey field c.13 3.13 2.9 1.86 3 10 2.7 300 All round 3 West Tofts 20 2.7 1.9 0.99 4 8.5 6.6 8000 W 4 Thorpe Gt heath c.9 2.95 2.2 1.32 3 7.7 7.7 2600 W 6 Military camp 10 3.91 2.8 1.44 3 7 3 2-400 SW & NE 7 Hockham 25 124.7 43 27.27 4 7 4 2-300 SW &NE 8 Peddars way adj. Poultry 16 16.46 9.2 5.63 3 5.3 0.5 50 E units 9 Peddars way adj. pigs c.22 9.81 4.54 1.4 3 10.3 2 50 All round 10a Norfolk Wildlife Reserve 2 12 4.45 1.6 1.92 2 7.2 3.5 800 SW 10b Norfolk Wildlife Reserve 3 15 2.91 1.9 1.12 2 7.2 5.5 1300 N & E 11 Stanford area c.19 2.03 1.4 0.86 3 10 6.1 7000 NW 12 Santon Downham FC 1 3.32 2.05 1.2 3 7.3 8 8000 W

Fig. 1. Map of Thetford area showing NH3 recording stations (see Appendix 1 for map of site with lichen stations).

# Thetford Study Area 19 Monitoring sites

21# #20

18# # # 27 # 26 25 9 # 10 # 17 # 12# 67## # 13 11# 16#

# 22 # # 23 24 15# 5# 1# 4# 8# 3# 2 # 14 N #

202468Kilometers

72 Lichen diversity A total of 45 lichen species were recorded in NH3 deposition and lichen diversity on oak trunks at Thetford the Thetford site with mean lichen numbers 11 varying from 4.7 to 10.7 species per station 10 9 (Appendix 1). The lowest diversity coincided 8 7

with proximity to intensive poultry units where per station 6 3 ammonia levels were highest (32-124 µg/m in lichens of Mean no. 5 4 October 2001) (fig. 2.). Overall the highest 012345678910 mean NH3 deposition ug/m3 diversity of lichens occurred on oaks on MOD land west of the poultry units in unimproved Fig. 2 grassland where stocking rates were low NH3 deposition and lichen diversity on twigs at (12,000 sheep on 27,000 acres (pers. Thetford y = -0.7847x + 6.9805 R2 = 0.5121 comm.)). Lichen diversity on twigs showed a 10 8 6 better correlation with ammonia deposition 4 2 per station than lichen diversity on trunks (figs. 2 & 3.). 0 Contrary to expectations, diversity was often Mean no. of lichens 012345678910 mean NH3 deposition ug/m3 highest on younger tree trunks and lowest on trees of great girth and age in all stations (fig. Fig. 3

4.), even on MOD land where there has been Tree girth vs trunk lichen diversity at Thetford no intensive agriculture for more than 50 450 14 400 12 350 years. This suggests previous damage to older 10 300 250 8 trunks probably from a long period of 200 6 150 4 Tree girth (cm) girth Tree 100 sustained acidification. Within the MOD area No lichen species 50 2 foliose species of the Parmelietum were 0 0 ts r of ath we e o T -l reserve st y present and often abundant in the canopy, but T e rpe h Hockham a Stanford W W NW Tho rs NWT langmereNWT ringmere Gt da d on the trunks they were absent or restricted to Pe the lowest parts of the tree trunk. Fig. 4 Diversity was also high in arable areas, although the community was dominated by NIW station values at Thetford 7 nitrophyte species of the Xanthorion. 6 5

W 4

NI 3 2 Distribution of nitrophyte and acidophyte 1 0 species. 012345678910 Only 9 NIW and 4 AIW species were recorded NH3 ug/m3 in the Thetford site in Norfolk from 20 possible Fig. 5 nitrophyte and 20 acidophyte species AIW station values at Thetford characterised by van Herk. Mean ammonia deposition in all stations adjacent to lichen 6 sampling was plotted against indices of AIW 4 AIW 2 and NIW for these stations (Figs. 5 & 6). The 0 NIW index showed a strong correlation with 012345678910 ammonia levels (Fig. 5), and the AIW showed NH3 ug/m3 little correlation with ammonia levels (Fig. 6). Fig. 6 However in stations adjacent to the highest ammonia deposition lichen diversity was 0 -1

73 on both trunks and twigs suggesting that there are critical loads of ammonia at which nitrophytes are absent. 3 Bark pH was also strongly affected in those areas where NH3 levels exceeded 10µg/m (Fig. 8). The stations in unimproved grassland in MOD or Forestry Commission sites with mean bark pH’s between 3 and 4.50 coincided with ammonia deposition below 3 (except site 12 where lichen sampling was in unimproved pasture upriver from the ammonia recording site which was by a horse-riding centre without oak trees). In contrast mean bark pH’s >5 were associated with stations where ammonia levels were above 10 µg/m3 (fig. 7). The local ammonia deposition gradient was confirmed by bark pH measurements at the Norfolk Wildlife’s Reserve (NWT), where bark pH was up to 6.2 in the vicinity of the poultry units and fell to c. 4 in the vicinity of Langmere 400m away. The directional nature of deposition was also indicated by variation in pH around the trunk on one young Quercus in the vicinity of the poultry unit, where a pH of 6.2 on the N. side of the trunk contrasted with a pH of 3.9 on the S side.

mean bark pH vs NH3 concentration (Thetford) Fig. 7 blue – trees in unimproved 10 grassland stations 5,11 & 12 with 8 low ammonia levels. 6 red – trees at stations 7,8 & 9 in the vicinity of high ammonia 4

NH3 (ug/m3) levels. 2 yellow – trees on NWT nature 0 reserve. 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 Bark pH

74 Site 2. North Wyke IGER, Devon. In Devon 7 sampling stations were studied at North Wyke (Fig. 8, Table 2.) and 77 lichen species recorded (see Appendix 2.).

Table 2. Stations sampled at North Wyke, Devon Lichen diversity Station No. Distance Directio trunk twig NIW AIW no trees from n from source source 2 5 -20 E 18.2 5 0.8 1 1 5 30 W 18 4.4 0.4 1.4 3 4 -300 E 15.7 9.3 0 0 4 4 300 W 20.7 11.5 1.25 3 5 3 750 NW 18.6 12.3 0 5 6 2 150 SW 16.5 0 1 7 4 700 W 18 11.2 0 3.5

Distribution of stations sampled at North Wyke shown in Appendix 2

Lichen diversity Lichen diversity per tree was high in all stations, averaging 17.6 on trees east of the cattle sheds and slurry tank to 20.7 on trees in pasture west of the sheds. Ammonia data was only available for one site to the west of the farm unit and levels were low throughout the year the highest being 3.54 µg/m3 during August and September in each year. As the main concentration of ammonia is from the overwintering cattle sheds and slurry tank east of the ammonia monitoring station, distance from source was used as a measure of likely ammonia distribution along an east to west transect. The prevailing wind is from the southwest (figs. 8 & 9).

Mean lichen diversity on Quercus trunks per station Lichen diversity on Quercu s trunks plotted against distance from source on an east west 25 20 axis shows little positive correlation (Fig. 8). Lichen 15 10 station diversity of twigs was considerably lower in the vicinity of 5

Mean diversity per 0 the cattle sheds and slurry tank and was positively -400 -200 0 200 400 600 800 1000 correlated with distance from source in both eastern and Distance from source (m) East to West western directions (Fig. 9). Fig. 8

This site also supports 10 lichen species that are Lichen diversity on Quercus twigs indicators of long ecological continuity (NIEC species) as 15 defined by Rose (1992), including the highly pollution- 10 sensitive Lobaria pulmonaria*, Teloschistes flavicans* station 5 0 and Usnea ceratina. These species occur in well-lit sites per mean diversity -400 -200 0 200 400 600 800 1000 with high relative humidity and absence of either Distance from source (m) East to West acidification or eutrophication (Table 3.). Other indicators Fig. 9 of long ecological continuity include Cresponea premnea and Lecanographa lyncea, both species characteristic of dry bark. These species were fragmented and in poor condition restricted to the densely 75 shaded drier E. side of the trunk. These ancient woodland indicators are now almost all restricted to a single tree on the boundary of the settlement (S6) and as such are relicts of a former wood pasture regime.

Table 3. Distribution of NIEC species in stations at North Wyke stations in order of distance from source

No. of trees at station 5 4 5 4 4 3 1 S2 S3 S1 S4 S7 S5 S6 Arthonia vinosa 2 Dimerella lutea 2 North Wyke NIW Cresponea lyncea 2 1.5

Lecanographa premnea 1 e 1 Lecanora jamesii 2 2 1 0.5 Lobaria pulmonaria 2 NIW valu 0 Punctelia reddenda 6 1 -400 -200 0 200 400 600 800 1000 Phaeographis dendritica 1 1 Distance from source (m) East to West Teloschistes flavicans 2 Fig. 10 Usnea ceratina 3 2 3 North Wyke AIW

y = 0.0043x + 1.1304 No of NIEC species 2 1 2 2 3 2 4 6 R2 = 0.9022 5 4 3 NIW and AIW distribution. AIW 2 1 12 AIW and 6 NIW species were recorded at North Wyke 0 -400 -200 0 200 400 600 800 1000 from a total of 20 AIW 20 NIW species as defined by van Distance from source (m) East to West Herk. However species of the Xanthorion in the NIW were rare or absent on trunks but present at low to high levels on Fig. 11 twigs at all sites, except in one sampling station on an acid wetland reserve where acidophytic species were dominant Bark pH of Quercus at stations, North Wyke 6 and the nitrophyte species of the Xanthorion absent. There 4 is little correlation between distance from the farm units and bark pH NIW species (Fig. 11) while AIW species show a strong 2 2 0 correlation – (R =0.9) with distance from source suggesting Station that the farm unit has an effect on these species. Fig. 12 Stations 1- orange, 2 - red, 3 - yellow, 4- pink, 5- blue, 7 - green Bark pH. Bark pH for all trees sampled showed little variation Bark pH v. distance from source North Wyke between trees at each station at North Wyke (Fig. 12). The 5.5 5 lowest bark pH occurred at station 5 on the edge of a

bark pH 4.5

Molinia moor. If bark pH is plotted against distance from 4 source, there is a trend towards higher pH in the vicinity of -400 -200 0 200 400 600 800 1000 Distance from source (m) East to West. the farm unit and a rapid fall off within 400m in both an Fig. 13 easterly and westerly direction (Fig. 13).This suggests that the farm unit is responsible for the observed changes in bark pH.

76 5. DISCUSSION

Variation between sites. Both sampling sites were on acid substrata, one on sand (Thetford) and the other on heavy riverine clays and loams (North Wyke), and both sites contained mature oak trees well distributed across the site. The sites are climatically very different. Rainfall in East Anglia averages 55 mm per year with considerable dry periods whereas in Devon the average is c. 176 mm per year with high relative humidity in all months. This allows a wide variety of oceanic lichen species to flourish and lichen cover and diversity tends to be higher in this region. Previous acidification effects are conspicuous in the Thetford region on the ancient oaks where lichen diversity was very low even in areas of unimproved wood pasture in the MOD area where there has been long ecological continuity. At North Wyke, where there is no evidence of former acidification, large old Quercus tree trunks support a highly diverse lichen community even in the vicinity of the farmstead including many oceanic species, pollution sensitive species and indicators of long ecological continuity (Table 1). In this site there was little impact of ammonia on long established natural lichen communities. Although both Thetford and North Wyke are areas with a tradition of wood pasture since Norman times with potentially long ecological continuity, the absence of former acidification and the higher rainfall at North Wyke must contribute to the survival of pollution sensitive species in this site.

Changes in temperature regimes may also contribute to changes in the distribution of lichen species. Many nitrophytes are predominantly warm temperate species and are benefiting from the trend of increasing mean annual temperatures associated with global warming (van Herk et al., 2002).

Nitrophyte and acidophyte species. Only 9 out of a possible 20 NIW species, and 8 out of 20 possible AIW species listed in van Herk were recorded in the surveys. This is partly due to the bias towards continental species in the Netherlands, and that the UK flora is more oceanic even in the eastern regions. Nitrophytes that are abundant in the Netherlands are absent or rare in Britain and others that may be considered as nitrophytes are excluded (see James et al. 1976). Alternative species considered to be nitrophytes in Britain that are present at the sites visited are indicated by a* in the table of species in the Appendices. Further testing in order to derive an appropriate species list for both nitrophytes and acidophytes is essential in regions where atmospheric conditions are monitored. In the Netherlands several new species have been described that are now appearing in Britain.

Ammonia deposition and lichens. Ammonia concentrations at stations in the area around Thetford were positively correlated with NIW indices and distance from source and showed little correlation between AIW indices and distance from source. There was no correlation of NIW species at North Wyke on trunks with distance from the farm unit as very few of the NIW species of the Xanthorion were present in the closed communities on the oak trunks. However, NIW species were present on twigs, indicating

77 that these species were colonising recently established lichen communities. At North Wyke ammonia levels were low, but the recording site was situated on the western side of the farm unit and would have only received higher levels during the distribution of slurry. The negative correlation of AIW species with distance from the farm unit, indicates that these acidophyte species are affected by the farm unit. The corresponding increase in bark pH in the vicinity of the farm unit correlates with the loss of acidophytic species characteristic of undisturbed lichen communities in this region. Bark pH correlated with lichen communities recorded at both Thetford and North Wyke and to the ammonia levels (Thetford) or distance from source (North Wyke). In areas where there is little environmental change, bark pH’s are relatively stable on the tree and at the site. Whereas in areas where changes are occurring as in the Thetford region, there are wide variations in bark pH even on one tree suggesting that there is a directional element in the pollutant causing an increase in bark pH. Increased bark pH is associated with an increase in ammonia deposition. Preliminary results on 3 trees at site 1 & 10 in the NWT reserve show a rapid change in bark pH suggesting that there is an environmental gradient on this site which agrees with Fowler et al. (1998) in falling off at c. 700m from the source. This is not supported by the ammonia deposition tubes which record highest levels of ammonia as 4.2µg/m3 in a recording station in the vicinity of station 10a. The positive correlation between ammonia deposition and NIW species at Thetford indicates a similarity between lowland sites in the east of Britain and the Netherlands. It also suggests that there are critically high levels of ammonia in this region where lichens including NIW species are absent (Figs 3 & 5).

Lichen diversity on trunks and twigs Preliminary observations from a basic inventory of lichen diversity on twigs at both sites suggest that primary colonisers of the newer bark surfaces are the most sensitive to changes in atmospheric conditions. In contrast, trunk species may reflect longer-term environmental conditions of ecological continuity (as at North Wyke) and former acidification through SO2 air pollution (Thetford). Although there was very little correlation between lichen diversity on trunks and proximity to sources of ammonia at North Wyke and Thetford, there was a strong correlation between lichen diversity on twigs and ammonia sources at both sites (figs. 3 & 9). Nitrophyte species were present in this community. At North Wyke nitrophyte Xanthorion species were conspicuously absent from the trunks but present on the twigs in the vicinity of the farmstead.

78 6. CONCLUSIONS

This pilot survey using techniques and indicator species defined as nitrophytes and acidophytes during research in the Netherlands (van Herk, 2002) suggests that epiphytic lichen communities on oak are sensitive to increased levels of atmospheric ammonia in Britain. However many NIW and AIW species recorded for the Netherlands were not found in sites sampled in either Norfolk or in Devon, suggesting that further work is needed to define appropriate nitrophyte and acidophyte indicator species in Britain. The research should define indicator species in relation to the wide climatic gradient across Britain, and in relation to other widespread nitrogen sources such as from fertilisers, car exhausts and fossil fuels, whose effect on lichen assemblages is poorly understood. Lichen species identified as possible candidates for these lists are indicated by a * in Appendices 3 & 4. As with the research in the Netherlands it is to be expected that species new to science or to Britain may be identified during surveys. The results of a rapid survey of lichens on twigs made during the pilot study is particularly interesting and suggests that there is a strong correlation between species colonising the new bark surface and distance from ammonia sources. Further research is needed together with NH3 monitoring to test the capacity for twig communities to respond to rapid fluctuations in NH3 concentrations over short periods of time, compared with communities and NIW/AIW indicators on trunks and twigs. The existence of a sampling method (Wolseley & Pryor, 1999; Wolseley, 2002) will allow a quantitative assessment of change in lichen communities to be tested against ammonia levels and other parameters.

7. RECOMMENDATIONS

• Define nitrophyte and acidophyte species appropriate for the UK based on multivariate analysis.

• Extend quantitative sampling to include twigs on oak trees across a gradient of NH3 deposition.

• Devise a practical recording method using lichens to detect changes in atmospheric nitrogen appropriate for Britain

• Include recording of bark pH together with ammonia and other nitrogen products monitoring across Britain, with particular reference to areas that were formerly acidified.

• Undertake further research on the effects of agricultural management on lichen communities including research stations where agricultural methods and eutrophication factors are being researched. The high lichen diversity at North Wyke makes this an ideal situation in which to extend ammonia monitoring and establish lichen monitoring.

79 APPENDIX A

Maps of lichen stations at Thetford and North Wyke.

80 APPENDIX B

Species identified by van Herk as nitrophytes and acidophytes (1999, 2002). Species highlighted were found during the survey.

Nitrophytes Acidophytes

Caloplaca citrina Cetraria chlorophylla C. holocarpa Chaenotheca ferruginea Candelaria aurella Cladonia sp. C. reflexa Evernia prunastri C. vitellina Hypocenomyce scalaris C. xanthostigma Hypogymnia physodes Lecanora muralis H. tubulosa L. dispersa grp. Lecanora aitema (incl. L. hagenii) L. conizaeoides Phaeophyscia orbicularis L. pulicaris P. nigricans Lepraria incana Physcia adscendens Ochrolechia microstictoides P. caesia Parmelia saxatilis P. dubia Parmeliopsis ambigua P. tenella Placynthiella icmalea Rinodina genarii Platismatia glauca Xanthoria candelaria Pseudevernia fufuracea X. calcicola Trapeliopsis flexuosa X. parietina T. granulosa X. polycarpa Usnea spp.

81 APPENDIX C. Table of species found on Quercus trunks in vicinity of Thetford with van Herk frequency values for each species at each station. Acidophytes in blue, nitrophytes in yellow. *Additional species to be considered as nitrophytes in the UK.

Order of stations by NH3 dep. 1 2 3 4 5 6 7 8 9 10 11 12 Mean diversity per trunk 7 5.3 10.3 10 7.4 7 7.7 6.1 7.5 8.5 10 7.3 Stations S7 S8 S9 S2 S1 S6 S4 S12 S10 S3 S11 S12 Species on trunk *Amandinea punctata 2 5 6 1 6 3 3 3 Arthonia spadicea 1 1 Arthonia sp 1 Athelia sp 1 *Bacidia delicata 1 2 2 2 2 Bryophytes 6 1 6 6 6 5 5 6 Candelariella reflexa 1 Candelariella vitellina 3 *Chrysothrix candelaris 2 Cladonia sp. 2 2 Cliostomum griffithii 2 3 1 6 3 2 3 3 Desmococcus 6 6 6 6 6 6 6 Dimerella pineti 1 1 Diploicia canescens 3 3 2 3 5 Evernia prunastri 2 1 6 6 3 2 *Hyperphyscia adglutinata 1 Hypogymnia physodes 2 cf Lecanora compallens 3 5 5 2 6 6 6 6 Lecanora dispersa 2 5 *Lecanora expallens 1 3 2 2 3 3 *Lecidella elaeochroma 2 1 Lepraria incana 5 2 6 6 6 6 6 6 Melanelia subaurifera 1 1 3 2 3 2 3 2 Micarea prasina 2 1 Haematomma caesium 2 Opegrapha atra 1 Flavoparmelia caperata 2 2 3 5 2 Hypotrachyna revoluta 2 Parmelia sulcata 1 6 3 2 2 Pertusaria amara 2 Pertusaria hymenea 2 Phaeophyscia orbicularis 2 Phlyctis argena 2 2 2 2 Physcia adscendens 2 1 5 Physcia tenella 2 5 6 6 5 3 2 3 *Physconia grisea 5 Punctelia borreri 1 Punctelia subrudecta 3 3 2 2 3 2 Punctelia ulophylla Pyrrhospora quernea 3 Ramalina farinacea 3 3 1 1 Ramalina fastigiata 3 Schismatomma decolorans 3 3 *Scoliciosporum chlorococcum 3 6 5 Xanthoria candelaria 3 2 1 3 2 3 3 Xanthoria parietina 6 2 6 6 3 3 2 2 Xanthoria polycarpa 3 2 3 5 2 1 2 2 Total 48 species 82

APPENDIX D

Table of species found on Quercus trunks at North Wyke, Devon with van Herk frequency values for each species at each station. Acidophytes in blue, nitrophytes in yellow. *Additional species to be considered as nitrophytes in the UK.

Stations sampled S1 S2 S3 S4 S5 S6 S7 Av. Species no. per station 18 17.6 18 20.7 18 18 16.5

Acrocordia conoidea 3 Acrocordia gemmata 2 *Amandinea punctata 5 4 3 3 Arthonia impolita 3 Arthonia radiata 2 2 Arthonia vinosa 2 *Bacidia delicata 2 3 Bacidia rubella 1 Candelariella reflexa 1 Candelariella vitellina 2 *Chrysothrix candelaris 5 2 3 3 4 Cladonia coniocreae 1 3 Cladonia pyxidata 2 Cladonia ramulosa 1 Cladonia sp. 2 3 1 Cliostomum griffithii 2 3 5 3 2 Dimerella lutea 2 Dimerella pineti 2 *Diploicia canescens 4 Enterographa crassa 1 4 2 Evernia prunastri 3 3 3 Fuscidea lightfootii 2 Hypogymnia physodes 1 2 Hypotrachyna revoluta 6 5 4 2 3 Cresponea lyncea 2 Lecanographa premnea 2 Lecanora chlarotera 5 3 4 5 3 2 3 Lecanora dispersa 1 *Lecanora expallens 6 6 6 6 6 2 6 Lecanora jamesii 2 2 1 *Lecidella elaeochroma 5 5 5 1 2 Lepraria sp 2 4 3 3 Lobaria pulmonaria 2 Melanelia fuliginosa 2 Melanelia subaurifera 5 3 3 3 Normandina pulchella 2 Ochrolechia subviridis 5 2 3 3 2 2 Ochrolechia turneri 1 Opegrapha atra 5 6 1 1 2 Opegrapha herbarum 3 Opegrapha multipuncta 1 Opegrapha vulgata Flavoparmelia caperata 6 4 1 5 3 6 Parmelia saxatilis 1 2 3 3

83 Parmelia sulcata 2 3 3 3 2 2 Parmotrema chinense 6 4 4 6 6 2 6 Pertusaria albescens v. corallina 4 3 6 6 2 6 Pertusaria albescens 2 6 2 1 Pertusaria amara 3 3 4 3 3 6 Pertusaria hemispherica 2 Pertusaria hymenea 6 6 6 6 6 2 5 Pertusaria pertusa 3 5 4 6 Phaeographis dendritica 1 1 Phaeophyscia orbicularis 1 Phlyctis argena 3 5 4 3 *Hyperphyscia adglutinata 3 1 *Physcia pulverea 3 Physcia tenella 2 Placynthiella icmalea 1 Punctelia borreri 1 Punctelia reddenda 5 1 Punctelia subrudecta 3 3 2 Punctelia ulophylla 3 Pyrrhospora quernea 5 5 1 3 6 6 Ramalina calicaris 1 Ramalina canariensis 2 Ramalina farinacea 2 1 4 3 4 Ramalina fastigiata 2 1 3 3 2 Rinodina roboris 1 Schismatomma cretaceum 3 Schismatomma decolorans 6 6 4 5 6 6 Teloschistes flavicans 2 Usnea ceratina 3 3 2 Usnea cornuta 3 2 2 Usnea flammea 2 3 Usnea rubicunda 1 1 Xanthoria parietina 2 2 1 Total diversity = 78 Total AIW = 12 3 3 0 5 9 1 5 Total NIW = 6 1 4 0 3 0 0 0

84 APPENDIX E

Table of species recorded on Quercus twigs Acidophytes in blue, nitrophytes in yellow.

North Wyke Thetford

Acrocordia conoidea Amandinea punctata Amandinea punctata Arthonia punctiformis Arthonia punctiformis Arthonia sp Arthonia radiata Arthopyrenia sp. Arthopyrenia fallax Athelia sp Arthopyrenia punctiformis Bacidia delicata Evernia prunastri Candelariella vitellina Fuscidea lightfootii Chrysothrix candelaris Graphina anguina Cliostomum griffithii Graphis scripta Desmococcus Hyperphyscia adglutinata Diploicia canescens Hypogymnia tubulosa Evernia prunastri Hypotrachyna revoluta Hyperphyscia adglutinata Lecanora albella Hypogymnia physodes Lecanora chlarotera Hypogymnia tubulosa Lecanora expallens Lecanora carpinea Lecanora intumescens Lecanora chlarotera Lecanora persimilis cf. Lecanora compallens Lecanora symmicta Lecanora expallens Lecidella elaeochroma Lecanora dispersa grp Lecidella elaeochroma f. soralifera Lecanora expallens Melanelia subaurifera Lecidea symmicta Micarea prasina Lecidella elaeochroma Opegrapha atra Lepraria incana Opegrapha herbarum Melanelia subaurifera Flavoparmelia caperata Mycoporum quercus Parmelia sulcata Flavoparmelia caperata Pertusaria amara Parmelia sulcata Phaeographis dendritica Pertusaria amara Physcia aipolea Phaeophyscia orbicularis Physcia tenella Physcia adscendens Punctelia borreri Physcia aipolea Punctelia subrudecta Physcia tenella Ramalina canariensis Phlyctis argena Ramalina farinacea Physconia grisea Ramalina fastigiata Punctelia borreri Scoliciosporum chlorococcum Punctelia subrudecta Usnea subfloridana Punctelia ulophylla Xanthoria parietina Ramalina farinacea Xanthoria polymorpha Scoliciosporum chlorococcum Xanthoria candelaria Xanthoria parietina Xanthoria polycarpa

85 REFERENCES

Farmer, A.M., Bates, J.W. & Bell, J.N.B. (1990) A comparison of methods for the measurement of bark pH. Lichenologist 22: 191-197 Fowler, D., Pitcairn, C.E.R., Sutton, M.a., Flechard, c., Loubet, B., Coyle, M. & Munro, R.C. (1998) The mass budget of atmospheric ammonia within 1km of livestock buildings. Environ. Pollut. (Nitrogen Conference Special Issue) 102, S1:343-348. James, P.W., Hawksworth, D.H. & Rose, F. (1977) Lichen communities in the British Isles. In Lichen Ecology. ed. Seaward, M.R.D.: 295-413. Academic press, London. NEGTAP 2001. Transboundary Air Pollution (2001) DEFRA. http://www.nbu.ac.uk/negtap/ Nimis. PL, Scheidegger, C. and Wolseley, P.A. (2002) Monitoring with Lichens – Monitoring Lichens. Nato Science Series, Kluwer, Dordrecht. 408 pp. Rose, F. (1992). Temperate forest management: its effects on bryophyte and lichen floras and habitats. In: Bryophytes and lichens in a changing environment. eds. Bates and Farmer: 211-233. Clarendon Press, Oxford. UK National Ammonia monitoring network http://www.nbu.ac.uk/cara/UKNAMN/uknamn.htm Van Herk, C.M. (1999) Mapping of ammonia pollution with epiphytic lichens in the Netherlands. Lichenologist 31: 9-20 Van Herk, C.M. (2002) Epiphytes on wayside trees as an indicator of eutrophication in the Netherlands. In Monitoring with Lichens – Monitoring Lichens. Eds Nimis. PL, Scheidegger, C. and Wolseley, P.A. Nato Science Series, Kluwer, Dordrecht. 285- 290. Van Herk, C.M., Aptroot, A. & van Dobben, H.F. (2002) Long-term monitoring in the Netherlands suggests that lichens respond to global warming. Lichenologist 34: 141-154 Wolseley, P.A. (2002) Using lichens on twigs to assess changes in ambient atmospheric conditions. . In Monitoring with Lichens – Monitoring Lichens. Eds Nimis. PL, Scheidegger, C. and Wolseley, P.A. Nato Science Series, Kluwer, Dordrecht. 291-294. Wolseley, P. A. & Pryor, K.V. (1999) The potential of epiphytic twig communities on Quercus petraea in a Welsh woodland site (Tycanol) for evaluating environmental changes. Lichenologist 31: 41-61.